Stabilized primate lentivirus envelope glycoproteins

ABSTRACT

A modified polypeptide corresponding to an envelope glycoprotein of a primate lentivirus is described. The polypeptide has been modified from the wild-type structure so that it has cysteine amino acid residues introduced to create disulfide bonds, a cavity is filled with hydrophobic amino acids, a Proresidue is introduced at a defined turn structure of the protein, or the hydrophobicity is increased across the interface between different domains, while retaining the overall 3-dimensional structure of a discontinuous conserved epitope of the wild-type protein. Preferably, the polypeptide has more than one of those characteristics. Preferably, the primate lentivirus is HIV, and the protein is HIV-1 gp120.

This application is a national stage entry under 35 U.S.C. § 371 ofinternational application PCT/US98/24001, filed Nov. 10, 1998, whichclaims benefit under 35 U.S.C. § 120 as a continuation-in-part of thefollowing U.S. applications: U.S. Ser. No. 08/966,932, filed Nov. 10,1997, now abandoned; U.S. Ser. No. 08/967,148, fled Nov. 10, 1997, nowabandoned; U.S. Ser. No. 08/967,708, filed Nov. 10, 1997, now abandoned;U.S. Ser. No. 08/967,403, filed Nov. 10, 1997, now abandoned; U.S. Ser.No. 08/966,987, filed Nov. 10, 1997, now abandoned; U.S. Ser. No.08/976,741, filed Nov. 24, 1997, now abandoned; U.S. Ser. No.09/100,762, filed Jun. 18, 1998, now abandoned; U.S. Ser. No.09/100,631, filed Jun. 18, 1998, now abandoned; U.S. Ser. No.09/100,521, filed Jun. 18, 1998, now abandoned; U.S. Ser. No.09/100,763, fled Jun. 18, 1998, now abandoned; U.S. Ser. No. 09/100,529,filed Jun. 18, 1998, now abandoned; and U.S. Ser. No. 09/100,764, filedJun. 18, 1998, now abandoned; and which claimed benefit under 35 U.S.C.§ 119(e) of U.S. provisional application 60/089,581, filed Jun. 17,1998, now abandoned, and U.S. provisional application 60/089,580, filedJun. 17, 1998, now abandoned.

FIELD OF THE INVENTION

The present invention is directed to envelope polypeptides having astructure that approximates conformational discontinuous epitopes of aprimate lentivirus envelope protein, but as a result of modifications ofthat structure has enhanced stability, raises a greater range ofantibodies to conserved epitopes, and/or has enhanced immunogenicity forbroadly neutralizing epitopes.

BACKGROUND OF THE INVENTION

Human immunodeficiency virus type 1 (HIV-1) is the cause of acquiredimmunodeficiency syndrome (AIDS), which is characterized by thedepletion of CD4-positive lymphocytes (See, Barre-Sinoussi, F., et al.,“Isolation of a T-lymphotropic Retrovirus From a Patient at Risk forAcquired Immunodeficiency Syndrome (AIDS),” Science 220:868–871 (1983);Gallo, R C, et al., “Frequent Detection and Isolation of CytopathicRetroviruses (HTLV-III) From Patients with AIDS and at Risk for AIDS,”Science 224:500–503 (1984)). Infection of humans by HIV-1 typicallyinvolves an initial period of acute, high-level viremia, followed by achronic, low-level viremia (See, Coombs, R W, et al., “Plasma Viremia inHuman Immunodeficiency Virus Infection,” N. Engl. J. Med. 321:1626–1631(1989); Clark, S J, et al., “High titers of Cytopathic Virus in Plasmafrom Patients with Symptomatic Primary HIV-1 Infection,” N. Engl. J.Med. 324:950–960 (1991); Daar, E S, et al., “Transient High Levels ofViremia in Patients with Primary immunodeficiency Virus Type 1Infection,” N. Engl. J. Med. 324:961–964 (1991); Fauci, A S, et al.,“Immunopathogenic Mechanisms of HIV Infection,” Ann. Inter. Med.124:654–663 (1996)). It is thought that the antiviral immune responsehelps to determine the “set-point” for chronic viremia. HIV-1persistence results in progressive CD4-positive lymphocyte decline,which ultimately compromises the immune response, including thatdirected against HIV-1. The resulting resurgence of high-level viremiais a harbinger of poor clinical outcome (See, Ho, DD, et al.,“Quantitation of Human Immunodeficiency Virus Type 1 in the Blood ofInfected Persons,” N. Engl. J. Med. 321:1621–1625 (1989)).

The envelope protein of a lentivirus is the most visible portion of thevirion because it is on the surface of the virus particle. Thus,considerable attention has focussed on the envelope protein as a targetfor inhibiting viral entry. Strategies that have been used include usingthe envelope protein to generate an immune response, decoys for theenvelope protein, etc. These approaches have not yet been successful.

It was recently reported that a large scale clinical trial was going tobe attempted with an HIV envelope protein as an immunogen. While theinitial trials with the protein have not been reported to be promisingin terms of showing any significant protective immunity, they have alsonot indicated any significant harm caused by the vaccine candidate. Thefact that a clinical trial with this type of preliminary results wouldbe attempted shows the importance placed upon the use of the envelopeprotein and underscores the need for improvements in enhancing theimmunogenicity of the envelope protein.

The envelope protein is an attractive target because, like that of otherretroviruses, the entry of HIV-1 into target cells is mediated by theviral envelope glycoproteins, gp120 and gp41, which are derived from agp160 precursor (See, Allan, J S, et al., “Major Glycoprotein AntigensThat Induce Antibodies in AIDS Patients are Encoded by HTLV-III,”Science 228:1091–1093 (1985); Robey, W G., et al., “Characterization ofEnvelope and Core Structural Gene Products of HTLV-III with Sera fromAIDS Patients,” Science 228:593–595 (1985)). The gp160 glycoprotein iscreated by the addition of N-linked, high mannose sugar chains to theapproximately 845–870 amino acid primary translation product of the envgene in the rough endoplasmic reticulum. Trimerization of gp160 in theendoplasmic reticulum is mediated by the formation of a coiled coilwithin the gp41 ectodomainu. (See, Earl, P L., et al., “OligomericStructure of the Human Immunodeficiency Virus Type 1 EnvelopeGlycoprotein,” Proc. Natl. Acad. Sci. USA 87:648–652 (1990); Pinter, A.,et al., “Oligomeric Structure of gp41, the Transmembrane Protein ofHuman Immunodeficiency Virus Type 1,” J. Virol. 63:2674–2679; Lu, M., etal., “A Trimeric Structural Domain of the HIV-1 TransmembraneGlycoprotein,” Nature Structural Biol. 2:1075–1082 (1995); Chan, D C, etal., “Core Structure of gp41 from the HIV Envelope Glycoprotein,” Cell89:263–273 (1997); and Weissenhorn, W., et al., “Atomic Structure of theEctodomain from HIV-1 gp41,” Nature 387:426–430 (1997)). The gp160trimers are transported to the Golgi apparatus, where cleavage by acellular protease generates the mature gp120 and gp41 glycoproteins,which remain associated through non-covalent interactions (Earl, P L, etal., “Folding, Interaction with GRP78-BiP, Assembly and Transport of theHuman Immunodeficiency Virus Type 1 Envelope Protein,” J. Virol.65:2047–2055 (1991); and Kowalski, M., et al., “Functional Regions ofthe Envelope Glycoprotein of Human Immunodeficiency Virus Type 1,”Science 237:1351–1355 (1987)). In mammalian host cells, addition ofcomplex sugars to selected, probably surface-exposed, carbohydrate sidechains of the envelope glycoproteins occurs in the Golgi apparatus.(See, Leonard, C K, et al., “Assignment of Intrachain Disulfide Bondsand Characterization of Potential glycosylation Sites of the Type 1Recombinant Human Immunodeficiency Virus Envelope Glycoprotein (gp120)Expressed in Chinese Hamster Ovary Cells,” J. Biol. Chem.265:10373–10382 (19990)).

Most of the surface-exposed elements of the oligomeric envelopeglycoprotein complex are contained on the gp120 exterior envelopeglycoprotein. (See, Moore, J., et al., “Probing the Structure of theHuman Immunodeficiency Virus Surface Glycoprotein gp120 with a Panel ofMonoclonal Antibodies,” J. Virol. 68:469–484 (1994)). When the gp120glycoproteins derived from different primate immunodeficiency virusesare compared, five conserved regions (C1 to C5) and five variableregions (V1 to V5) can be identified. (See, Starcich, B R, et al.,“Identification and Characterization of Conserved and Variable Regionsof the Envelope Gene HTLV-III/LAV, the Retrovirus of AIDS,” Cell45:637–648 (1986); Myers, G., et al. “Human Retroviruses and AIDS: ACompilation and Analysis of Nucleic Acid and Amino Acid Sequences,” LosAlamos National Laboratory, (1994)). Intramolecular disulfide bonds inthe gp120 glycoprotein result in the incorporation of the first fourvariable regions into large, loop-like structures. Antibody bindingstudies and deletion mutagenesis have indicated that the major variableloops are well-exposed on the surface of the gp120 glycoprotein. (See,Wyatt, R., et al., “Functional and Immunologic Characterization of HumanImmunodeficiency Virus Type 1 Envelope Glycoproteins ContainingDeletions of the Major Variable Regions,” J. Virol. 67:4557–4565 (1993);Pollard, S., et al., “Truncated Variants of gp120 bind CD4 with HighAffinity and Suggest a Minimum CD4 Binding Region,” EMBO J. 11:585–591(1992)).

The mature envelope glycoprotein complex is incorporated into HIV-1virions, where it mediates virus entry into the host cell. The gp120exterior glycoprotein binds the CD4 glycoprotein, which serves as theprimary receptor. (See, Klatzmann, D., et al., “T-lymphocyte T4 MoleculeBehaves as the Receptor for Human Retrovirus LAV,” Nature London312:767–768 (1984); and Dalgleish, A G., et al., “The CD4 (T4) Antigenis an Essential Component of the Receptor for the AIDS Retrovirus,”Nature 312: 763–767 (1984)). The association of gp120 with CD4 ismediated by the interaction of a discontinuous gp120 structure with theCDR2-like region of the CD4 amino-terminal domain. (See, Brodsky, M H.,et al., “Analysis of the Site in CD4 that Binds to the HIV EnvelopeGlycoprotein,” J. Immunol. 144: 3078–3086 (1990); Peterson, A., et al.,“Genetic analysis of Monoclonal Antibody and HIV binding Sites on theHuman Lymphocyte Antigen CD4,” Cell 54:65–72 (1988); Moebius, U., etal., “The Human Immunodeficiency Virus gp120 Binding Site on CD4:Delineation by quantitative Equilibrium and Kinetic Binding Studies ofMutants in Conjunction with a High-Resolution CD4 Atomic Structure,” J.Exp. Med. 176:507–517 (1982); Arthos, J., et al., “Identification of theResidues in Human CD4 Critical for the binding of HIV,” Cell 57:469(1989); Ryu S E., et al., “Crystal Structure of an HIV-bindingRecombinant Fragment of Human CD4,” Nature London 348:419–425 (1990);and Wang, J., et al., “Atomic Structure of a Fragment of Human CD4containing Two immunoglobulin-like Domains,” Nature London 348:411–418(1990)). Amino acids in the gp120 C3 and C4 regions have been implicatedin CD4 binding. (See, Cordonnier, A., et al., “Single Amino Acid Changesin HIV Envelope Affect Viral Tropism and Receptor Binding, Nature340:571–574 (1989); Lasky, L., et al., “Delineation of a Region of theHuman Immunodeficiency Virus Type 1 gp120 Glycoprotein Critical forInteraction with the CD4 Receptor,” Cell 50:975–985 (1987); andOlshevsky, U., et al., “Identification of Individual HIV-1 gp120 AminoAcids Important for CD4 Receptor Binding,” J. Virol. 64:5701–5707(1990)). The association of gp120 with CD4 is believed to initiateconformational changes in the HIV-1 envelope glycoprotein complex,leading to interactions with members of the chemokine receptor family.(See, Sattentau, Q., et al., “Conformational Changes Induced in theHuman Immunodeficiency Virus Envelope Glycoprotein by Soluble CD4binding,” J. Exp. Med. 174:407–415 (1991); Thali, M., et al.,“Characterization of Conserved Human Immunodeficiency Virus Type 1(HIV-1) gp120 neutralization Epitopes Exposed Upon gp120-CD4 Binding,” JVirol 67:3978–3988 (1993); Sattentau, Q., et al., “ConformationalChanges Induced in the Envelope Glycoproteins of Human and SimianImmunodeficiency Virus by Soluble Receptor Binding,” J. Virol.67:7388–7393 (1993); Trkola, A., et al., “CD4-dependent,antibody-sensitive Interactions Between HIV-1 and its Co-receptorCCR05,” Nature 384:184–187 (1996); and WU, L., et al., “CD4-inducedInteraction of Primary HIV-1 gp120 Glycoproteins with the ChemokineReceptor CCR5,” Nature 384:179–183 (1996).

Chemokine receptors are G protein-coupled, seven-membrane-spanningproteins involved in leukocyte chemotaxis. (See, Baggioline, M., et al.,“Interleukin-8 and Related Chemotactic Cytokines-CXC and CC Chemokines,”Adv. Immunol. 55:97–179 (1994); Gerard, N., et al., “thePro-Inflammatory Seven-Transmembrane-Segment Receptors of theLeukocyte,” Curr. Opin. Immunol. 6:140–145 (1994); and Premack, B A., etal, “Chemokine Receptors: Gateways to Inflammation and Infection,”Nature Medicine 11:1174–1178 (1996)). Most laboratory-adapted HIV-1viruses utilize a CXC chemokine receptor called CXCR4 (also calledLESTR, HUMSTSR or fusin), while most macrophage-tropic primary HIV-1viruses use the CC chemokine receptor CCR5 (see, Feng, Y., et al.,Science 272:872–877 (1996); Choe, H. et al., Cell 85:1135–1148 (1996);Deng. H K., et al., Nature 381:661–666 (1996); Dragic, T., et al.,Nature 381:667–673 (1996); Doranz, B J., et al., Cell 85:1149–1158(1996); and Alkhatib, G., et al., Science 272:1955–1958 (1996)), and toan extent CCR3 or CCR2. Primary dual-tropic HIV-1 isolates use CCR5 aswell as CXCR4. (See, Zhang, L., et al., Nature 383:768 (1996) andConnor, R., et al., J. Exp. Med. 185:21–628 (1997)). Themacrophage-tropic primary viruses are those most often transmitted frominfected to uninfected individuals, and predominate during the long,asymptomatic phase of infection. (See, Cheng-Mayer, C., et al., Science240:80–82; Zhu, T., et al., Science 261:1179–1181 (1993); Fenyo, E., J.Virol. 62:4414–4419 (1988); Schuitemaker, H., et al., J. Virol.66:1354–1360 (1991); and Connor, R I, et al., J. Virol. 67:1772–1778(1993)). The importance of CCR5 for HIV-1 transmission is underscored bythe observation that humans with homozygous defects in CCR5 arerelatively resistant to HIV-1 infection. (See, Liu, R., et al., Cell86:367–378 (1996); Samson, M., et al., Nature 382:722–725 (1996); andDean M., et al., Science 273:1856–1862 (1996)). CCR5 is used as acorrector by almost all primary HIV-1 isolates regardless of geographicclade, and is used by the related human and primate immunodeficiencyviruses, HIV-2 and simian immunodeficiency virus, SIV. (See, Marcon, L.,et al., J. Virol 71:2522–2527 (1997); Chen, Z., et al., J. Virol.71:2705–2714 (1997); and Cocchi, F., et al., Science 270:1811–1815(1995)). This suggests that at least part of the viral binding site forCCR5 is well-conserved among these immunodeficiency viruses. While thesegp120 structures are under investigation and have yet to be completelydefined, mutagenic studies have suggested that elements of the V3 loopmay constitute part of the chemokine receptor binding site. Geneticstudies of viruses with chimeric HIV-1 envelope glycoproteins containingdifferent V3 loops demonstrated that the gp120 V3 region is a majordeterminant of which chemokine receptor, CCR5 or CXCR4, can be used asan entry cofactor. (See, Cocchi, F., et al., Nature med., 2:1244–1247(1996); and Speck, R., et al., J. Virol. (in press)). Thus, even in therelatively variable background of the V3 domain, there may existconserved structural features that collaborate with other conservedgp120 structures to create a high-affinity binding site for CCR5.

It is likely that the interaction of the gp120-CD4 complex with theappropriate chemokine receptor promotes additional conformationalchanges in the envelope glycoprotein complex. By analogy with theinfluenza hemoglutinin, it has been suggested that the HIV-1 gp41ectodomain undergoes major conformational changes during virus entry.(See, Carr, C M., et al., Cell 73:823–832 (1993); Chen, C H., et al., J.Virol. 69:3771–3777 (1995); Bullough, P., et al. Nature 371:37–43(1994); and Weissenhorn, W., et al., EMBO J. 15:1507–1514 (1996)). Theproposed result of these changes is the insertion of the hydrophobicgp41 amino terminus (the “fusion peptide”) into the membrane of thetarget cell. Mutagenic analysis and the recently determined crystalstructures of HIV-1 gp41 ectodomain fragments are consistent with thismodel (see, Freed, E., et al., Proc. Natl. Acad. Sci USA 87:4650–4654(1990)).

The exposed nature of the HIV-1 envelope glycoproteins on the surface ofvirions or infected cells renders them prime targets for the antiviralimmune response. In fact, the only viral proteins accessible toneutralizing antibodies are the envelope glycoproteins. Neutralizingantibodies appear to be an important component of a protective immuneresponse, in chimpanzees challenged with HIV-1 (see, Berman, P W., etal., Nature 345:622–625 (1990); Girard, et al., Proc. Natl. Acad. Sci.USA 88:542–546 (1991); Emini, et al., Nature 355:728–730 (1991); andBruck, et al., Vaccine 12:1141–1148 (1994). That neutralizing antibodiesgenerated during the course of HIV-1 infection do not provide permanentantiviral effect may in part be due to the generation of neutralizationescape virus variants (see, Nara, et al., J. Virol. 64:3779–3791 (1990);Gegerfelt, et al., Virology 185:162–168 (1991); and Arendrup, et al., JAIDS 5:303–307 (1992)), and to the general decline in the host immunesystem associated with pathogenesis.

HIV-1 neutralizing antibodies are mostly directed against linear ordiscontinuous epitopes of the gp120 exterior envelope glycoprotein. Rareexamples of gp41-directed neutralizing antibodies have also beendocumented (see, Muster, et al., J. Virol. 67:6642–6647 (1993)).Neutralizing antibodies that arise early in infected humans and that arereadily generated in animals by immunization are primarily directedagainst linear neutralizing determinants in the third variable (V3) loopof gp120 glycoprotein (see, Matthews, et al., Proc. Natl. Acad. Sci. USA83:9709–9713 (1986); and Javaherian, et al., Science 250:1590–1593(1990)). These antibodies generally exhibit the ability to neutralizeonly a limited number of HIV-1 strains, although some subsets of anti-V3antibodies recognize less variable elements of the region and thereforeexhibit broader neutralizing activity (see, Ohno, et al., Proc. Natl.Acad. Sci. USA 88:10726–10729 (1991); Moore, et al., J. Virol.69:122–133 (1995); and Gorny, et al., J. Virol. 66:7538–7542 (1992)).Envelope glycoprotein variation within the linear V3 epitope and outsideof the epitope can allow escape of viruses from neutralization by theseantibodies (see, McKeating, et al., J. Virol. 67:4932–4944 (1993)). Thesecond variable (V2) region of the HIV-1 envelope glycoprotein has alsobeen shown to be a target for strain-restricted neutralizing antibodies(see, Fung, et al., J. Virol. 66:848–856 (1992); Moore, et al., J.Virol. 67:6136–6151 (1993)). Most of the V2 epitopes consist ofcontinuous but conformation-dependent determinants.

Later in the course of HIV-1 infection of humans, antibodies capable ofneutralizing a wider range of HIV-1 isolates appear (see, Profy, et al.,J. Immunol. 144:4641–4647 (1990); Berkower, et al., J. Em. Med. 170:1681–1695 (1989); Ho, et al., J. Virol. 489–493 (1991); Kang, et al.,Proc. natl. Acad. Sci. USA 88:6171–6175 (1991); Steimer, et al., Science254:105–108 ((1991); and Moore et al., J. Virol. 67:863–875 (1993)).These broadly-neutralizing antibodies have been difficult to elicit inanimals (see, Rusche et al., Proc. Natl. Acad. Sci. USA 84:6924–6928(1987); Klaniecki et al., AIDS Res. Hum. Retro. 7:791–798 (1991); andHaigwood, et al., J. Virol. 66:172–182 (1992)), and are not merely theresult of additive anti-V3 loop reactivities against diverse HIV-1isolates that accumulate during active infection. A subset of thebroadly reactive antibodies, found in most HIV-1-infected individuals,interferes with the binding of gp120 and CD4. At least some of theseantibodies recognize discontinuous gp120 epitopes (the so-called CD4BSepitopes) present only on the native glycoprotein. Human monoclonalantibodies derived from HIV-1-infected individuals have been identifiedthat recognize the gp120 glycoproteins from a diverse range of HIV-Iisolates, that block gp120-CD4 binding, and that neutralize virusinfection (see, Posner, et al., J. Immunol. 146:4325–4332 (1991); andTilley, et al., Res. Virol. 142:247–259 (1991)). Some of theseCD4BS-directed antibodies efficiently neutralize primary HIV-1 isolates(see, Burton, et al., Science 266:1024–1027 (1994)), which are generallymore resistant to neutralization than are viruses passaged inimmortalized cell lines (see, Daar, et al., Proc. Natl. Acad. Sci. USA87:6574–6578 (1990); Wrin, et al., J. virol. 69:39–48 (1995); Sullivan,et al., J. Virol. 69:4413–4422 (1995); Sawyer, et al., J. Virol.67:1342–1349 (1994); Moore, et al., J. Virol. 69:101–109 (1995); andD'Souza, et al., J. Infect. Dis. 175:(in press)(1997)). Thediscontinuous epitopes recognized by many of the human monoclonalantibodies directed against the CD4BS epitopes have been characterizedby mutagenic analysis (see, Thali, et al., J. Virol. 65:6188–6193(1991); Thali, et al., J. Virol. 66:5635–5641 (1992); McKeating, et al.,Virology 190:134–142 (1992)). Amino acid changes in seven discontinuousgp120 regions, four of which overlap regions defined to be important forCD4 binding, disrupt recognition by these antibodies and, in some cases,allow the generation of neutralization escape mutants.

A second group of neutralizing antibodies found in a smaller number ofHIV-1-infected humans is directed against conserved gp120 epitopes thatare exposed better upon CD4 binding (see, Thali, et al., J. Virol.67:3978–3988 (1993)). These epitopes, referred to as the CD4-induced(CD4i) epitopes, are extremely sensitive to conformational changes inthe gp120 glycoprotein. The integrity of these epitopes is affected bygp120 amino acid changes in the conserved stem of the V1/V2 stem-loopstructure and in the C4 region. The CD4i epitopes have been shown to beproximal to the V3 loop and to be masked by the V1/V2 variable loops(see, Wyatt, et al., J. Virol. 69:5723–5733 (1995); and Moore, et al.,J. Virol 70:1863–1872 (1996)). It has been shown that CD4 bindinginduces a movement of the V 1/V2 loops that exposes the CD4i epitopes.Interestingly, it has been shown that neutralizing antibodies directedagainst either the V3 loop or the CD4i epitopes block the ability ofgp120-CD4 complexes to bind CCR5. Thus, it appears that the major groupsof neutralizing antibodies generated in HIV-1-infected humans block thebinding of virus to its cellular receptors., either CD4 or the chemokinereceptors.

The development of an HIV-1 vaccine as explained above has been hamperedby the inefficiency with which antibodies directed against the moreconserved gp120 structures are elicited. Most of the antibodies elicitedby the HIV-1 envelope glycoproteins, either in infected humans or chimpsor in animals immunized with envelope glycoprotein preparations, are notable to neutralize virus. Many of these non-neutralizing antibodies aredirected against gp120 structures that are inaccessible on the nativeenvelope glycoprotein complex due to interaction with the gp41ectodomain (see, Wyatt, et al., (1997)). When neutralizing antibodiesare elicited, these are often directed against variable portions of theHIV-1 envelope glycoproteins. Most of the neutralizing antibodieselicited by native HIV-1 gp120 or gp160 glycoproteins are directedagainst the V3 loop (see, Haigwood, et al., AIDS Res. Hum. Retro.6:855–869 (1990)). Multiple immunizations with native gp120 or gp160glycoproteins are required to elicit even low titers of neutralizingantibodies with broader strain reactivity. This same pattern ofelicitation of neutralizing antibodies has been observed inHIV-1-infected humans or chimps, with antibodies directed against the V3loop appearing earlier in infection. These results suggest that thestructure of the HIV-1 gp120 envelope glycoprotein has evolved todecrease the immunogenicity of particular epitopes in which variation ispoorly tolerated by the virus. By the time immune responses to theseepitopes are elicited, immune compromise has occurred, viral burden ishigh, and virus variation and the potential for neutralization escapehas reached significant levels. These considerations suggest that use ofthe native, complete HIV-1 glycoprotein as an immunogen will mostefficiently elicit the same types of immune responses that the virus hasevolved to evade most efficiently. Improved immunogens based upon theenvelope protein are necessary.

Previous studies have indicated that the relatively poor surfaceaccessibility of the more conserved gp120 epitopes related to the CD4and chemokine receptor binding sites may in part provide an explanationfor the low apparent immunogenicity of these regions.

One approach to improve the immunogenicity of gp120 polypeptides hasbeen to remove at least a portion of the “masking” variable loops whileretaining the overall conformation of the polypeptide so that itapproximates that of the native gp120. This can be done by appropriateselection of amino acid residues to permit the structure to turn. Inthis manner the conserved conformational epitopes are more exposed andcan be used to generate antibodies to these conserved epitopes.Additional improvements in generating such polypeptides would be useful.The V1/V2 and V3 variable loops of the HIV-1 gp120 glycoprotein havebeen shown to mask the CD4BS epitopes, and removal of these variableregions results in a 5-50-fold increase in exposure of most of the CD4BSepitopes, on both the monomeric and the multimeric envelopeglycoproteins. Removal of the V1 and V2 variable loops results in anincreased exposure of HIV-1 gp120 epitopes (V3 and CD4i epitopes)located near the binding site for the chemokine receptors. Thus, both ofthe receptor-binding regions of the HIV-1 gp120 glycoprotein arepartially masked by the large variable loop structures of theglycoprotein.

It is imperative that means of efficiently eliciting an array ofantibodies directed against the more conserved gp120 elements bedeveloped.

SUMMARY OF THE INVENTION

We have now found polypeptides that are modified from a primatelentivirus envelope glycoprotein such as the HIV-1 envelopeglycoproteins that can improve the stability and/or enhanceimmunogenicity of neutralization epitopes, particularly those conservedon different primary viruses such as the CD4BS and/or CD4i epitopes. Themodifications include the deletion of particular variable loops and/orstabilization of functionally relevant envelope glycoprotein structuresthrough the formation of internal disulfide bonds. For example, we havefound that introducing cysteine residues at at least one of thefollowing pairs of amino acid residues results in the formation ofdisulfide bonds and substantially stabilizes the structure of theprotein:

-   -   Pro 118*--Ala 433    -   Leu 122--Gly 431    -   Phe 210--Gly 380    -   Ser 256--Phe 376    -   The numbering is based upon HXBc2 numbering and can readily be        extrapolated to other viruses and strains.        Preferably disulfide bonds are introduced at either        Pro118→Ala433, Leu122-Gly431, Phe210-Gly380, or Ser256-Phe376.

Alternatively, or in addition, one can fill the cavities discovered inthe interior of HIV-1 gp120 with hydrophobic residues such asSer375→Trp, Val155→Trp, Arg273→Trp, Ser481→Phe, Ser447→Ile. Thesecavity-filling substitutions should stabilize a native HIV-1 gp120conformation.

Alternatively, or in addition, one can introduce prolines at definedturn structures such as Ile423→Pro, thus stabilizing these turnstructures in the gp120 “bridging sheet,” which appear to beconformationally flexible (see below).

Alternatively, or in addition, one can increase the hydrophobicityacross the interface between the gp120 domains such as Asn377→Leu,Thr283→Ile, and Asp477→Leu. These substitutions are predicted todecrease interdomain flexibility.

These changes can be inserted in a polypeptide that contains all thevariable regions, or more preferably, into a polypeptide wherein atleast a portion of a variable region, preferably the V1/V2 loops, hasbeen deleted with a linker amino acid residue inserted to retain turnsin the structure so that it approximates the conformation of at leastone discontinuous conformation epitope of the native envelope proteinsuch as CD4BS or CD4i epitopes.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A–1E show the structure of the HIV-1 gp120 region implicated inCCR5 binding.

FIG. 1A shows a ribbon drawing of the HIV-1 gp120 glycoprotein complexedwith CD4. The perspective is that from the target cell membrane. The twoamino-terminal domains of CD4 are shown in blue. The gp120 inner domainis colored red, the outer domain is colored yellow, and the “bridgingsheet” is orange. The gp120 residues in which changes resulted in a ≧90%decrease in CCR5 binding are labeled. The V1/V2 stem and the base of theV3 loop (strands β12 and β13 and the associated turn) are indicated.

FIG. 1B shows a molecular surface of the gp120 glycoprotein from thesame perspective as that of FIG. 1A. Colored surfaces are associatedwith gp120 residues in which changes resulted in either a ≧75% decrease(yellow), a ≧90% decrease (red) or a ≧50% increase (green) in CCR5binding, when CD4 binding was at least 50% of that seen for the wtΔprotein.

FIG. 1C shows the surface depicted in FIG. 1B colored according to thedegree of conservation observed among primate immunodeficiency viruses(25). Red indicates conservation among all human and simianimmunodeficiency viruses; orange indicates conservation among all HIV-1isolates, including group O and chimpanzee isolates; yellow indicatesmodest variability and green indicates substantial variability amongHIV-1 20 isolates.

FIG. 1D shows the molecular surface of the gp120 glycoprotein,indicating residues in which changes resulted in a ≧70% decrease in 17bantibody binding, in the absence of sCD4.

FIG. 1E shows the molecular surface of the gp120 glycoprotein,indicating residues in which changes resulted in a ≧70% decrease in CG10antibody binding in the presence of sCD4. Residues in which changessignificantly decreased CD4 binding (and thus indirectly decreased CG10binding) are not shown. Images were made with Midas-Plus (ComputerGraphics Lab, University of California, San Francisco) and GRASP.

FIG. 2 shows the molecular surface of the gp120 outer domain coloredaccording to the variability observed in gp120 residues among primateimmunodeficiency viruses. Red indicates residues conserved among allprimate immunodeficiency viruses; orange, residues conserved in allHIV-1 isolates; yellow, residues exhibiting some variation among HIV-1isolates; and green, residues exhibiting significant variability amongHIV-1 isolates. The inner gp120 domain is colored red and the outerdomain is colored yellow. The Bridging sheet” is colored orange. The N-and C-termini of the truncated gp120 core are labeled, as are thepositions of structures related to the gp120 variable regions, V1–V5.The HA, TIC, ED and HE surface loops 2 are shown. The position of the“Phe 43” cavity involved in CD4 binding is indicated by an asterisk. Agp120 surface implicated in binding to the CCR5 chemokine receptor isindicated. The variability of the gp120 surface shown is underestimatedsince the V4 variable loop, which is not resolved in the structure,contributes to this surface (approximate location is indicated). Theposition of the V5 region is shown. Also note the highly conservedglycosylation site (asparagine 356 and threonine/serine 358) within theHE loop, between the V5 and V4 regions. In the figure on the right, theV4 loop and the carbohydrates are modeled, as described in Materials andMethods. The complex carbohydrate addition sites used in mammalian cells4 are colored light blue, and the high-mannose sites are colored darkblue. The gp120 protein surface is shown in white.

FIGS. 3A–3D show the spatial relationship of epitopes on the HIV-1 gp120glycoprotein.

FIG. 3A shows the molecular surface of the gp120 core The modeledN-terminal gp120 core residues, V4 loop and carbohydrate structures areincluded. The variability of the molecular surface is indicated, usingthe color scheme described in FIG. 2. The modeled carbohydrates arecolored light blue (complex sugars) or dark blue (high-mannose sugars).The approximate locations of the V2 and V3 variable loops are indicated.Note the well-conserved surfaces near the “Phe 43” cavity and thechemokine receptor-binding site.

FIG. 3B shows a Ca tracing of the gp120 core. The gp120 residues within4 Å of the 17b CD4i antibody are shown in green. The residues implicatedin the binding of CD4BS antibodies20 are shown in red. Changes in theseresidues significantly affect the binding of at least 25 percent of theCD4BS antibodies listed in Table 1. The residues implicated in 2G12bindings are shown in blue. The V4 variable loop, which contributes tothe 2G12 epitope, 9 is indicated by dotted lines.

FIG. 3C shows the molecular surface of the gp120 core, oriented andcolored as in Figure B.

FIG. 3D shows the approximate locations of the faces of the gp120 core,defined by the interaction of gp120 and antibodies. The molecularsurface accessible to neutralizing ligands (CD4 and CD4BS, CD4i and 2G12antibodies) is shown in white. The neutralizing face of the completegp120 glycoprotein includes the V2 and V3 loops, which reside adjacentto the surface shown. The approximate location of the gp120 face that ispoorly accessible on the assembled envelope glycoprotein trimer andtherefore elicits only non-neutralizing antibodiesS6 is shown in purple.The approximate location of an immunologically “silent” face of gp120,which roughly corresponds to the highly glycosylated outer domainsurface, is shown in blue.

FIG. 4 is a schematic showing the probably arrangement of the HIV-1gp120 glycoproteins in a trimeric complex. The gp120 core was organizedinto a trimeric array, based on the criteria discussed in the text. Theperspective is from the target cell membrane, similar to that shown inFIG. 2. The CD4 binding pockets are indicated by black arrows, and theconserved chemokine receptor-binding regions are colored red. The areasshaded light green indicate the more variable, glycosylated surfaces ofthe gp120 cores. The approximate locations of the 2G12 epitopes areindicated by blue arrows. The approximate locations for the V3 loops(yellow) and V4 regions (green) are shown. The positions of the V5regions (green) and some complex carbohydrate addition sites(asparagines 276, 463, 356, 397 and 406) (blue dots) are shown. Theapproximate locations of the large V1/V2 loops, centered on the knownpositions of the V1/V2 stems, are indicated (green). On one of the gp120subunits, the positions of the ID and HE loops are indicated. Thedistance

DETAILED DESCRIPTION OF THE INVENTION

We have discovered a series of novel polypeptides that can (1) enhancethe immunogenicity of primate lentivirus envelope proteins for certainconserved epitopes, (2) generate a greater range of antibodies against“masked” gp120 structures and/or (3) stabilize the three-dimensionalstructure of the molecule.

We have discovered regions where disulfide bonds can be inserted whichwill stabilize the conformation of the molecule in a conformationapproximating the native envelope glycoprotein conformation. We havediscovered conserved regions and epitopes that are critical for CD4 andchemokine receptor binding. We have discovered critical turn structuresof the molecules as well as internal cavities that decrease theimmunogenicity of epitopes that would raise antibodies that could blockCD4 binding and/or chemokine binding.

Preferably, the envelope protein is selected from the group consistingof HIV or SIV. More preferably, it is HIV. Still more preferably, it isHIV-1 gp120.

We have succeeded in growing crystals of gp120 (from the HXBc2 HIV-1strain) in a ternary complex with two-domain CD4 (D 1 D2 sCD4) and theFab fragment of a CD4i neutralizing antibody, 17b Fab. The crystalsdiffracted to a minimum Bragg spacing of at least 2.2A, and data havebeen collected from cryogenically preserved crystals on the nativecomplex as well as on isomorphous heavy atom derivatives. While someelements of the HIV-1 gp120 structure (e.g. the V3 loop) are notsupplied by analysis of these crystals, the vast majority of the gp120residues are able to be defined in the structure. Importantly, all ofthe gp120 residues thought to contribute to the CD4BS and CD4ineutralization epitopes are defined in the available structure.

Many of the antibody responses elicited against the HIV-1 envelopeglycoproteins during natural infection of humans are incapable ofneutralizing the virus. Studies of monoclonal antibodies derived fromHIV-1-infected individuals indicate that most of these non-neutralizingantibodies are directed against elements of the gp120 and gp41glycoproteins that interact on the assembled oligomer. These elementsare not accessible on the functional envelope glycoprotein spike on thevirus membrane or infected cell surface, thereby rendering theantibodies directed against them ineffectual at neutralization. Thelabile association of gp120 and gp41, which exposes and/or creates theepitopes for these non-neutralizing antibodies, apparently represents anadaptive mechanism for lentiviruses such as HIV-1 to divert the humoralimmune response under conditions where antigen is limiting.

A corollary is that the gp120 glycoprotein dissociated from thefunctional oligomer may have evolved to be less effective at elicitingneutralizing antibodies directed against conserved gp120 structures.This corollary appears to be supported by the many attempts to elicitneutralizing antibodies by gp120 immunogens over the past several years.Dissociation from gp41 apparently results in an increase in theconformational flexibility of the gp41-interactive regions of gp120,predisposing the gp120 glycoprotein to elicit non-neutralizingantibodies preferentially over the more broadly neutralizing antibodies.This conformational flexibility can have two consequences relevant toselective elicitation of non-neutralizing antibodies:

1) The flexibility and surface exposure of the gp41-interactive C1 andC5 regions on free gp120 can make these structures more immunogenic; and

2) Conformational flexibility in the C 1 and C5 regions, can mask manyCD4BS epitopes, may disrupt these epitopes and decrease the efficiencywith which CD4BS-directed antibodies are elicited.

Thus, we have found a number of positions where disulfide bonds can beintroduced to stabilize the polypeptide's structure. This is importantgiven the structure of the molecule.

For example, the gp120 core is composed of an inner domain, an outerdomain, and a “bridging sheet” (FIG. 1A). The “bridging sheet” is afour-stranded, antiparallel β-sheet that includes the V1/V2 stem andstrands (β20 and β21) derived from the fourth conserved gp120 region.CD4 contacts gp120 residues in the outer domain and the “bridgingsheet”. The gp120 residues implicated by our study in CCR5 binding arelocated near or within the “bridging sheet” (FIGS. 1A and 1B). The“bridging sheet” is predicted to face the target cell after the envelopeglycoproteins bind CD4. Even more than the CD4-binding site, the gp120region implicated in CCR5 binding is highly conserved among primateimmunodeficiency viruses; this is particularly apparent in comparison tothe remainder of the gp120 surface thought to be exposed on theassembled envelope glycoprotein complex (See FIGS. 1C and 2). The CD4iepitope for the 17b antibody is located near or within the “bridgingsheet”, consistent with the ability of the antibody to block CCR5binding. All of the individual gp120 residues in which changes disruptedrecognition by the 17b antibody (FIG. 1D) are located close to thegp120-17b interface in the crystallized complex (Table 1). The bindingof another antibody, CG10, which disrupts gp120-CCR5 interaction andcompetes with the 17b antibody for gp120 binding, is also affected bychanges in amino acid residues within or near the “bridging sheet” (FIG.1E). The position and orientation of the V3 base in the structure, inconjunction with a number of mutagenic and antibody competition studies,indicates that the gp120 V3 loop resides proximal to the regionimplicated in CCR5 binding (FIG. 1A). For example, the binding of bothCG10 and CD4i antibodies to gp120 can be disrupted by some V3 changes.Furthermore, several V3-directed antibodies compete with CD4i antibodiesfor gp120 binding.

We have discovered that the CCR5-binding site is likely composed ofconserved gp120 elements near or within the “bridging sheet” and V3 loopresidues. The latter apparently includes more conserved structures (e.g.the aromatic or hydrophobic residue at position 317), as well as morevariable structures that determine the specific chemokine receptor used.Some of the gp120 residues identified in this and previous studies asdeterminants of chemokine receptor utilization can modulate theinteraction of the V3 loop and elements near the “bridging sheet”.Studies of HIV-1 revertants suggested a functional interaction of gp120residue 440, shown here to influence CCR5 binding, with the V3 loop.

A subset of the gp120 residues in or near the “bridging sheet”apparently contacts CCR5 directly. Most of the gp120 residues implicatedin CCR5 binding exhibit reasonable solvent accessibility in the freegp120 core (Table 1). The gp120 surface implicated in CCR5 binding ishighly basic, favoring interactions with the acidic CCR5 amino terminus,which has been shown to be important for gp120 binding. Additional,hydrophobic interactions, similar to those seen for gp120-17b binding,can also contribute to the gp120-CCR5 interaction.

The exposure and/or formation of the CCR5-binding site of HIV-1 gp120glycoproteins is dependent upon interaction with CD4. CD4 binding hasbeen shown to reposition the V1/V2 variable loops and thus expose theCD4i epitopes, which overlap the CCR5-binding region. However, since agp120 glycoprotein lacking the V1 and V2 variable loops also exhibitsCD4-dependent CCR5 binding, the interaction with CD4 must cause otherconformational changes in gp120 related to the CCR5-binding site. Ourresults, which highlight the proximity of the two receptor-binding siteson gp120, help explain the induction of such conformational changes.First, one of the components of the “bridging sheet”, the V1/V2 stem,also contacts CD4. Thus, CD4 binding, which appears to distort the V1/V2stem, may reposition this structure and allow the formation of thep-sheet important for CCR5 binding. In this respect, a substitution ofaspartic acid for threonine 123, which is located in the V1/V2 stem andcontacts CD4, significantly decreases CCR5 binding. This substitutioncan disrupt CD4-induced conformational changes in the V1/V2 stemrequired for CCR5 binding. Second, the CD4-bound conformation of gp120exhibits a cavity (the “Phe 43” cavity) within the gp120 interior. Thiscavity contacts the gp120 inner and outer domains as well as the“bridging sheet” and likely forms as a result of interdomainconformational changes in gp120 induced by CD4 binding. Since the“bridging sheet” lacks its own hydrophobic core and is thus dependentupon residues contributed by both inner and outer domains, any shift inorientation between these domains would alter the conformation of the“bridging sheet”. Furthermore, CD4 binding could also alter the preciseorientation of the “bridging sheet” with respect to the inner and outerdomains, thus aligning the V3 loop and conserved gp120 elementsimportant for CCR5 binding.

CD4 binding induces conformational changes within the “bridging sheet”as well as between this sheet and the inner and outer domains to formthe high-affinity CCR5 binding site. For some primate immunodeficiencyviruses, the CD4-bound conformation of gp120 must be energeticallyassessable in the absence of CD4, which would explain the documentedexamples of CD4-independent chemokine receptor binding and entry.

The CCR5-binding region defined in this study using HIV-1 is alsoimportant for the binding of the other primate lentiviruses such assimian, and of human immunodeficiency viruses to other chemokinereceptors. The identified region exhibits one of the most highlyconserved surfaces on the HIV-1 gp120 glycoprotein, supporting itsfunctional importance for all primate immunodeficiency viruses. Thelaboratory-adapted HXBc2 envelope glycoprotein, which uses CXCR4 and notCCR5 as a corrector, can be converted to an efficient CCR5-using proteinsimply by substituting the V3 loop of the YU2 virus. Thus, all of thenecessary CCR5-binding region outside of the V3 loop are conserved, asdemonstrated by the substitution between the divergent HXBc2 and YU2viruses. Indeed, we have shown that alteration of the lysine 117, lysine207 and glycine 441 in the HXBc2-YU2V3 chimeric protein also disruptsCCR5 binding. Consistent with the use of this region for the binding ofother chemokine receptors is the observation that the gp120 changesassociated with the conversion of HIV-2 to a CD4-independent,CXCR4-using virus affect the “bridging sheet” and the V3 loop.Alterations in “bridging sheet” residues have also been implicated inchanges in the tropism of HIV-1 for immortalized cell lines that do notexpress CCR5. And, the 17b antibody neutralizes HIV-1 strains that usedifferent chemokine receptors, thereby supporting our finding of theinvolvement of a common gp120 region in chemokine receptor interaction.

Chemokine receptor binding can trigger additional conformational changesin the envelope glycoprotein complex that ultimately lead to the fusionof the viral and target cell membrane. Some of these changes includeexposure of the ectodomain of the gp41 transmembrane envelopeglycoprotein. The CCR5-binding region defined herein resides close tothe trimer axis of the assembled envelope glycoprotein complex. Indeed,some of the gp120 residue changes that affect CCR5 binding also affectthe non-covalent association of gp120 and gp41 subunits in the trimericcomplex. This indicates that chemokine receptor binding alters therelationship between gp120 and gp41, leading to the exposure of the gp41ectodomain and interaction with the target cell membrane.

Stabilizing the structure of an envelope protein such as the gp120glycoprotein should improve the ability of the glycoprotein to elicitdesirable neutralizing antibody responses. This follows from ourobservation that all of the conserved HIV-1 gp120 neutralizationepitopes span gp120 domains that exhibit potential flexibility.Stabilization of the gp120 structure can be achieved by introducing newdisulfide bridges at specific locations on the gp120 chain. Thistargeted introduction of disulfides is designed to maintain the moleculein a conformation wherein at least the CD4BS or CD4i epitopesapproximate the wild-type conformation. We expect this disulfide bondingto preserve the integrity of the relevant neutralization epitopes.

The disulfide bonds can be introduced at a number of different aminoacid residues in the gp120 structure. The only precaution is not toreplace an amino acid residue critical for the generation of an antibodyto a conserved epitope. A number of these epitopes are set forth in thetables generated by the binding assay. Residues that can be used includePro118-Ala433 (using HXBc2 numbering), Leu122-Gly431, Phe210-Gly380, andSer256-Phe376. The respective amino acid residues in other strains canreadily be derived by standard means such as aligning the amino acidsequence by any standard computer homology program (e.g. these include,but are not limited to BLAST 2.0 such as BLAST 2.0.4 and 2.0.5 availablefrom the NIH (See www.ncbi.nlm.nkh.gov/BLAST/newblast.html) (Altschul,S. F., et al. Nucleic Acids Res. 25: 3389–3402 (1997))and DNASIS(Hitachi Software Engineering America, Ltd.) under the default setting.Preferably one inserts disulfide bonding at one of Pro-Ala or Leu-Glyand one of Phe-Gly or Ser-Phe.

In addition, other residues that can be used can be determined basedupon the following criteria:

1) The two residues targeted for cysteine substitution are distant onthe gp120 linear sequence, thus increasing the entropic benefit of thecysteine bridge (see below);

2) The C atoms of the selected residues are within 6× of one another andthe C_(β), atoms within 4× of each other, in the native gp120 structure;

3) Neither of the selected residues is proximal in the structural modelto naturally occurring gp120 cysteines, nor do natural disulfide bondsalready link the targeted gp120 strands;

4) The substituted residues as aforesaid, do not make majorcontributions to the binding of desired neutralizing antibodies;

5) If internal residues are chosen, both residues are involved in mutualpacking interactions.

Adherence to these criteria should optimize the opportunity to generatewell-folded gp120 glycoprotein derivatives in which the naturaldisulfide bonds form and the introduced cysteines create an additionalnovel disulfide bond. Within a 6× inter-C distance, the possibility foreither cis- or trans-disulfide bond formation allows considerableflexibility in interatomic distances.

These choices can be further confirmed by taking the overall energyconsiderations into account. For example, theoretical and empiricalstudies of the effects of added covalent cross-links on the folded statehave been conducted on model proteins (see, Hazes, et al., Protein Eng.2:119–125 (1988); Muskai et al., Protein Eng. 3:667–672 (1990); Reiter,et al., Protein Eng. 8:1323–1331 (1995); Sowdhamini, et al., ProteinEng. 3:95–103 (1989); Zhou, et al., Biochem 32:3178–3187 (1993);Johnson, et al., Biochem. 17:1479–1483 (1978); and Pace, et al., J.Biol. Chem. 263:11820–11825 (1988)). Most proteins can be modeled asexisting in two states, native and unfolded, the ratio of which at anygiven temperature, pH and salt concentration can be specified by anequilibrium constant K_(F) (see. Kyte, et al., Structurei n ProteinChemistry pp. 445–466 (1995)). The equilibrium constant of folding(K_(F)) is related to the standard free energy of folding ( )G°_(F)) bythe equation)G°_(F)=RT in K_(F), where R is the gas constant and T isthe temperature. The)G° F. value is primarily the sum of the favorableenthalpic contribution of removal of hydrophobic amino acids fromcontact with the aqueous environment and the unfavorable loss ofconfigurational entropy of the unfolded, random coil. Under physiologicconditions, the)G° F. value for most proteins is slightly negative (−30to −60 kJ/mole), thus favoring the native conformation.

The introduction of disulfide or other covalent bonds cross-linkingstrands of a protein has been demonstrated to stabilize the native stateof the protein, lowering the)G° F. value. Since proteins must be alreadyfolded to allow cysteines that are adjacent in the native structure toform a disulfide bond, and cysteine bridges per se contribute little toenthalpic changes favorable to folding, the vast majority of thestabilizing effect of disulfide bonds on the native state derives from adecrease in the configurational entropy of the unfolded protein. Apractical consequence of this is that the greater the distance in thelinear amino acid sequence between the two cysteines that arecross-linked, the greater the magnitude of the stabilizing effect on thenative conformation. These theoretical considerations have beensupported by experiments introducing cross-links into proteins atvarious positions and determining the resulting K_(F) and)G°_(F) values.The decreases in)G° F. associated with cross-linking in theseexperiments were on the order of −20 kJ/mole, which can exertconsiderable effects on stabilization of native structure (consideringthat the difference between unfolded and native status is typically only−30 to −60 kJ/mole). Since the existing intrachain disulfide bridges inthe HIV-1 gp120 glycoprotein only minimally constrain the potentialconformations available to the denatured protein, a significant benefitshould accrue by introducing additional, properly positionedcross-links.

The gp120 exists in three domains, and the presence of cavities wedgedbetween these domains offers the possibility of interdomain flexibility.Since the conserved neutralization epitopes on gp120 span two domains,such flexibility can render the protein incapable of efficientlyeliciting these kinds of desirable antibodies. The selective use ofintroducing hydrophobic amino acid residues in the modified envelopeprotein can enhance immunogenicity as discussed below.

The disulfide stabilized mutants can be created by the site-directedmutagenesis of a plasmid designed to express the soluble HIV-1 gp120glycoprotein in the supernatants of Drosophila cells by known means. Forexample, 89.6 and YU2 gp120 or any other gp120 glycoproteins can beused. Cell supernatants can be examined for the production of properlyfolded gp120 glycoproteins, using a pool of sera from HIV-1-infectedhumans, which will recognize even misfolded gp120 molecules, and a panelof conformation-dependent anti-gp120 monoclonal antibodies. Properlyfolded proteins with desirable epitopes intact will be purified byimmunoamnity chromatography using a CD4BS-directed antibody (F105)column.

Several methods are available to document the formation of, for example,the desired disulfide bond in the gp120 glycoprotein. Chemical methodsallow an estimate of the percentage of the proteins in a givenpreparation that form the disulfide bond. For example, ethyleniminereacts with cysteine under mild conditions to form anS-(_(β)aminoethyl)-cysteine derivative, which can be detected in proteinhydrolysates by chromatographic analysis. The presence of thesederivatives indicates that at least some of the cysteines in the proteinare free, and the percentage of these unpaired cysteines can beestimated by using other methods that do not distinguish cysteine fromcystine (e.g., ethylenimine in conjunction with a reducing agent, orperformic acid oxidation (Rafferty, Biochem. Biophys. Res. Commun.10:467 (1963); and Moore, S., J. Biol. Chem. 238:235 (1963)). Analysisof proteolytic fragments of the wild-type and mutant glycoproteins is asecond approach capable of documenting the formation of the desireddisulfide bond. The latter method can be used in conjunction withmonoclonal antibodies directed against specific linear peptides of gp120to verify that peptides in the vicinity of the putative disulfide bondexhibit altered behavior upon proteolysis of wild-type and mutantglycoproteins.

The formation of an additional disulfide bond bridging linearly distantgp120 regions that are not already constrained by existing disulfidebonds should result in a significant effect on K_(F) and)G° F. Sinceunder physiological conditions, most proteins are stably folded in theirnative state, estimates of K_(F) and)G°_(F) are typically made underconditions of low pH, higher temperature and/or the presence of urea orguanidinium chloride. Since the protein folding reaction must occurreversibly to obtain estimates of K_(F) or)G°_(F), the test should avoidthe use of high temperatures that often lead to irreversible changes inproteins. Instead, the denaturation of the wild-type and cross-linkedmutant gp120 glycoproteins should be compared over a range of chaotropicsalt concentrations and pH values. A number of physical properties ofproteins have been used to monitor protein folding, including intrinsicviscosity, optical rotation, molar ellipticity, ultraviolet lightabsorption, electrophoretic mobility and sedimentation velocity.Absorption of ultraviolet light can be studied for the wild-type andmutant gp120 glycoproteins produced in Drosophila cells, since thisparameter is easily measured and reliably detects changes in proteinfolding. The two states of gp120, native and denatured, exist, K_(F)and)G° F. can be determined for each concentration of guanidiniumchloride, temperature and pH directly from the absorbance versus salt/pHcurves. Typically, K_(F) and)G° F. values obtained under these varyingconditions are used to extrapolate to physiologic salt and pH values,although the stabilizing effect of the introduced disulfide should beevident over a wide range of pH and chaotropic salt concentrations.

As mentioned above, one can also alternatively introduce Pro at definedturn structures. For example, at Ile423. These changes can readily bemade and tested, specifically to see that the integrity of relevantneutralization epitopes is retained.

To enhance the ability to generate antibodies one can increase thehydrophobicity of various cavities in the molecule. The presence ofcavities in the CD4-bound gp120 structure probably reflects interdomainflexibility in the non-CD4-bound portion. The interdomain flexibilitycould decrease the integrity of CD4BS and CD4i epitopes, and otherconserved structures.

One way of dealing with this problem is to increase the hydrophobicresidues in the cavity. Hydrophobic residues are well-known and includeTrp, Phe, Leu, and Ile. One can change some of the non-hydrophobicresidues into hydrophobic residues, or increase the hydrophobicity ofalready hydrophobic residues. An increase in the size of the side chaincan be tolerated, depending on the volume of the cavity to be filled.The changes can be made by site-directed mutagenesis or other knownmeans. The changes can be tested for their effect on antibody binding byusing a panel of known antibodies that bind to a desired epitope, e.g.,using CD4BS epitopes. Examples of the changes that can be made includeSer375→Trp, Val255→Trp, Arg273→Trp, Ser481→Phe, and Ser447→Ile.Preferably, at least one of the amino acid residues in the cavity arechanged, i.e., they are Trp or Phe or Ile, instead of the wild-typeconfiguration.

The recessed nature of the CD4 binding pocket may delay the generationof high affinity antibodies against the CD4BS epitopes and can affordopportunities to minimize the antiviral efficacy of such antibodies oncethey are elicited. The degree of recession is believed to be evengreater on the full length glycosylated gp120 than is evident on thecrystallized gp120 core. The recessed pocket is flanked on one side bythe V1/V2 stem loop structure. The V2 loop apparently folds back alongthe V1/V2 stem with V2 residues 183–188 proximal to Asp 368 and Glu 370.This can enhance masking of the adjacent CD4BS and CD4i gp120 epitopesand divert antibody responses toward the variable loops. This may bedealt with by using gp120 polypeptides where at least a portion of thevariable loop has been deleted as described in U.S. Pat. No. 5,817,316.

Still more preferably, more than one of the amino acid residues havethese changes.

One can also increase the hydrophobicity across the interface betweenthe gp120 domains. Hydrophobic residues that fill the interdomaincavities will decrease interdomain flexibility.

Thus, one should increase the generation of antibodies by the conservedreceptor-regions, and can enhance immunogenicity or raise a greaternumber of antibodies to these desired sites than the wild-type proteindoes. This can be done by having the polypeptide contain hydrophobicresidue at certain interface sites instead of other residues. Forexample, having Leu, Ile, Trp, etc., such as Leu instead of Asn377, Ileinstead of Thr283, and/or Leu instead of Asp477. The key to thesubstitution is to preserve the conformational integrity of the desiredneutralization epitope, while at the same time filling the interdomaincavities.

The integrity of relevant neutralization epitopes on an envelopeglycoprotein such as gp120 can be verified with a panel of monoclonalantibodies, as described above. Purified mutant proteins that exhibitformation of e.g., the desired disulfide bond and increased stability ofa native conformation can be used to immunize mice, in parallel with thewild-type gp120 as a control.

The polypeptides of this invention can be used to generate a range ofantibodies to gp120. For example, antibodies that affect the interactionwith the binding site can be directly screened for example using adirect binding assay. For example, one can label, e.g. radioactive orfluorescent, a gp120 protein or derivative and add soluble CD4. Thereare various soluble CD4s known in the art including a two-domain (D1D2sCD4) and a four-domain version. The labeled gp120, or derivative, e.g.,a conformationally intact deletion mutant such as one lacking portionsof the variable loops (e.g. V1/V2) and in some instances constantregions and soluble CD4 can be added to medium containing a cell lineexpressing a chemokine receptor that the antibody will block binding to.In this example, the derivative will block binding to CCR5.Alternatively, when using a derivative from a T cell tropic gp120 onewould use a cell line that expresses CXCR4. Binding can then be directlymeasured. The antibody of interest can be added before or after theaddition of the labeled gp120 or derivative and the effect of theantibody on binding can be determined by comparing the degree of bindingin that situation against a base line standard with that gp120 orderivative, not in the presence of the antibody.

A preferred assay uses the labeled gp120, or derivative portion, forexample a gp120 protein derived from an M-tropic strain such as JR-FL,iodinated using for instance solid phase lactoperoxidase (in one examplehaving a specific activity of 20 μCi/μg). The cell line containing thechemokine receptor in this example would be a CCR5 cell line, e.g. L1.2or membranes thereof. Soluble CD4 would be present.

In one embodiment, the conformational envelope polypeptide, such asgp120 should contain a sufficient number of amino acid residues todefine the binding site of the gp120 to the chemokine receptor (e.g.typically from the V3 loop) and a sufficient number of amino acids tomaintain the conformation of the peptide in a conformation thatapproximates that of wild-type gp120 bound to soluble CD4 with respectto the chemokine receptor binding site. Preferably, the V1/V2 loops aredeleted. In other embodiments at least portions of the V3 loop can beremoved to remove masking amino acid residues. In order to maintain theconformation of the polypeptide one can insert linker residues thatpermit potential turns in the polypeptides structure. For example, aminoacid residues such as Gly, Pro and Ala. Gly is preferred. Preferably,the linker residue is as small as necessary to maintain the overallconfiguration. It should typically be smaller than the number of aminoacids in the variable region being deleted. Preferably, the linker is 8amino acid residues or less, more preferably 7 amino acid residues orless. Even more preferably, the linker sequence is 4 amino acid residuesor less. In one preferred embodiment the linker sequence is one residue.Preferably, the linker residue is Gly.

In one preferred embodiment, the gp120 also contains a CD4 binding site(e.g. from the C3 region residues 368 and 370, and from the C4 regionresidues 427 and 457). The chemokine binding site is a discontinuousbinding site that includes portions of the C2, C3, C4 and V3 regions. Bydeletion of non-essential portions of the gp120 polypeptide—such asdeletions of portions of non-essential variable regions (e.g. V1/V2) orportions in the constant regions (e.g. C1, C5) one can increase exposureof the CD4 binding site. Another embodiment is directed to a gp120portion containing a chemokine binding site. Similarly, by deleting thenon-essential portions of the protein one can increase exposure of thechemokine binding site. The increased exposure enhances the ability togenerate an antibody to the CD4 receptor or chemokine receptor, therebyinhibiting viral entry. Removal of these regions is done while requiringthe derivative to retain an overall conformation approximating that ofthe wild-type protein with respect to the native gp120 binding region,e.g. the chemokine binding region when complexed to CD4. In addition,one can remove glycosylation sites that are disposable for properfolding (see Wyatt et al., U.S. provisional application no.EL014417278US, filed Jun. 17, 1998). Maintaining conformation can beaccomplished by using the above-described linker residues that permitpotential turns in the structure of the gp120 derivative to maintain theoverall three-dimensional structure. Preferred amino acid residues thatcan be used as linker include Gly and Pro. Other amino acids can also beused as part of the linker, e.g. Ala. Examples on how to prepare suchpeptides are described more fully in Wyatt, R., et al. J. of Virol.69:5723–5733 (1995); Thali, M., et al., J. of Virol. 67:3978–3988(1993); and U.S. Pat. No. 5,817,316, issued Oct. 6, 1998 which areincorporated herein by reference. See for example Wyatt which teacheshow to prepare V1/V2 deletions that retain the stem portion of the loop.

In one embodiment the gp120 derivative is designed to be permanentlyattached at the CD4 binding site to sufficient domains of CD4 to createa conformation of the chemokine binding site approximating that of thenative gp120 CD4 complex.

An alternative gp120 derivative is one wherein the linkers used resultin a conformation for the derivative so that the discontinuous bindingsite or a discontinuous epitope such as CD4BS or CD4i with the chemokinereceptor approximates the conformation of the discontinuous binding sitefor the chemokine receptor in the wild-type gp120/CD4 complex. Thesederivatives can readily be made by the person of ordinary skill in theart based upon the above described methodologies and screened in theassays shown herein to ensure that proper binding is obtained.

The gp120 polypeptide can also be bound to at least a portion of a gp41polypeptide, namely the coiled coil. Some of these derivatives will lackthe gp41 transmembrane region and will therefore be made as secreted,soluble oligomers. For example, gp41 portions lacking the transmembraneregion but retaining the cytoplasmic region, others truncated beginningwith the transmembrane region. The gp41 polypeptide may containadditional cysteine residues, which result in the formation of the SHbonds between the monomers thereby stabilizing the complex as a trimerhaving spikes similar to that found in the wild type (as in U.S.application Ser. No. 09/164,880).

These immunogenic oligomers can be used to generate an immune reactionin a host by standard means. For example one can administer thepolypeptide in adjuvant. In another approach, a DNA sequence encodingthe envelope protein, e.g., the one based upon gp120 can be administeredby standard techniques. The approach of administering the protein ispresently preferred.

The protein is preferably administered with an adjuvant. Adjuvants arewell known in the art and include aluminum hydroxide, Ribi adjuvant,etc. The administered protein is typically an isolated and purifiedprotein. The protein is preferably purified to at least 95% purity, morepreferably at least 98% pure, and still more preferably at least 99%pure. Methods of purification while retaining the conformation of theprotein are known in the art. The purified protein is preferably presentin a pharmaceutical composition with a pharmaceutically acceptablecarrier or diluent present.

DNA sequences encoding these proteins can readily be made. For example,one can use the native gp 160 (or a derivatized gp120 portion) of any ofa range of primate lentiviruses such as HIV-1 strains which are wellknown in the art and can be modified by known techniques such asdeleting the undesired regions such as variable loops and to insertdesired coding sequences such as cysteines and linker segments. Inaddition to DNA sequences based upon existing strains, the codons forthe various amino acid residues are known and one can readily preparealternative coding sequences by standard techniques.

DNA sequences can be used in a range of animals to express the monomer,which then forms into the trimer and generates an immune reaction.

DNA sequences can be administered to a host animal by numerous methodsincluding vectors such as viral vectors, naked DNA, adjuvant assistedDNA catheters, gene gun, liposomes, etc. In one preferred embodiment theDNA sequence is administered to a human host as either a prophylactic ortherapeutic treatment to stimulate an immune response, most preferablyas a prophylactic. One can administer cocktails containing multiple DNAsequences encoding a range of HIV env strains.

Vectors include chemical conjugates such as described in WO 93/04701,which has targeting moiety (e.g. a ligand to a cellular surfacereceptor), and a nucleic acid binding moiety (e.g. polylysine), viralvector (e.g. a DNA or RNA viral vector), fusion proteins such asdescribed in PCT/US 95/02140 (WO 95/22618) which is a fusion proteincontaining a target moiety (e.g. an antibody specific for a target cell)and a nucleic acid binding moiety (e.g. a protamine), plasmids, phage,etc. The vectors can be chromosomal, non-chromosomal or synthetic.

Preferred vectors include viral vectors, fusion proteins and chemicalconjugates. Retroviral vectors include moloney murine leukemia virusesand HIV-based viruses. One preferred HIV-based viral vector comprises atleast two vectors wherein the gag and pol genes are from an HIV genomeand the env gene is from another virus. DNA viral vectors are preferred.These vectors include herpes virus vectors such as a herpes simplex Ivirus (HSV) vector [Geller, A. I. et al. J. Neurochem 64: 487 (1995);Lim, F. et al., in DNA Cloning: Mammalian Systems, D. Glover, Ed.(Oxford Univ. Press, Oxford England) (1995); Geller, A. I. et al., ProcNatl. Acad. Sci. U.S.A. 90: 7603 (1993); Geller, A. I., et al., ProcNatl. Acad. Sci USA 87: 1149 (1990)], adenovirus vectors [LeGal LaSalleet al., Science 259: 988 (1993); Davidson, et al., Nat. Genet 3: 219(1993); Yang, et al., J. Virol. 69: 2004 (1995)] and adeno-associatedvirus vectors [Kaplitt, M. G., et al., Nat. Genet. 8:148 (1994)].

The DNA sequence can be operably linked to a promoter that would permitexpression in the host cell. Such promoters are well known in the artand can readily be selected. For example, when expression in a mammalianhost is desired, a promoter that results in high levels of expression insuch host cells is used. Appropriate polyalkenylation sequences are alsoknown and can be selected. Representative examples of such promoters,include a retioviral LTR or SV40 promoter, the E. coli. lac or trp, thephage lambda P[L]promoter and other promoters known to controlexpression of genes in prokaryotic or eukaryotic cells or their viruses.The expression vector also contains a ribosome binding site fortranslation initiation and a transcription terminator. The vector mayalso include appropriate sequences for amplifying expression.

Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are pKK232-8 and pCM7. Particular namedbacterial promoters include lac, lacZ, T3, T7, gpt, lambda P[R], P[L]andtrp. Eukaryotic promoters include CMV immediate early, HSV thymidinekinase, early and late SV40, LTRs from retrovirus, and mousemetallothionein-I. Selection of the appropriate vector and promoter iswell within the level of ordinary skill in the art.

In a further embodiment, the present invention relates to host cellscontaining the above-described constructs. The host cell can be a highereukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell,such as a yeast cell, or the host cell can be a prokaryotic cell, suchas a bacterial cell. Introduction of the construct into the host cellcan be effected by a variety of methods including calcium phosphatetransfection, DEAE-Dextran mediated transfection, or electroporation(Davis, L., Dibner, M., Battey. I., Basic Methods in Molecular Biology,(1986)).

Stabilized forms of these complexes can readily be made, for example, byconjugates such as a poly(alkylene oxide) conjugate. The conjugate ispreferably formed by covalently bonding the hydroxyl terminals of thepoly(alkylene oxide) and a free amino group in the gp120 portion thatwill not affect the conformation of the discontinuous binding site.Other art recognized methods of conjugating these materials includeamide or ester linkages. Covalent linkage as well as non-covalentconjugation such as lipophilic or hydrophilic interactions can be used.

The conjugate can be comprised of non-antigenic polymeric substancessuch as dextran, polyvinyl pyrrolidones, polysaccharides, starches,polyvinyl alcohols, polyacryl amides or other similar substantiallynon-immunogenic polymers. Polyethylene glycol(PEG) is preferred. Otherpoly(alkylenes oxides) include monomethoxy-polyethylene glycolpolypropylene glycol, block copolymers of polyethylene glycol, andpolypropylene glycol and the like. The polymers can also be distallycapped with C1–4 alkyls instead of monomethoxy groups. The poly(alkyleneoxides) used must be soluble in liquid at room temperature. Thus, theypreferably have a molecular weight from about 200 to about 20,000daltons, more preferably about 2,000 to about 10,000 and still morepreferably about 5,000.

One can administer these stabilized compounds to individuals by avariety of means. For example, these antibodies can be included invaginal foams or gels that are used as preventives to avoid infectionand applied before people have sexual contact.

The peptides or antibodies when used for administration are preparedunder aseptic conditions with a pharmaceutically acceptable carrier ordiluent.

Doses of the pharmaceutical compositions will vary depending upon thesubject and upon the particular route of administration used. Dosagescan range from 0.1 to 100,000 μg/kg a day, more preferably 1 to 10,000μg/kg.

Routes of administration include oral, parenteral, rectal, intravaginal,topical, nasal, ophthalmic, direct injection, etc.

Changes in the viral envelope glycoproteins, in particular in the thirdvariable (V3) region of the gp120 exterior envelope glycoprotein,determine tropism-related phenotypes (Cheng-Mayer et al., 1990; O'Brienet al., 1990; Hwang et al., Westervelt et al., 1992; Chesebro et al.,1992; Willey et al., 1994). Amino acid changes in the V3 region (Helsethet al., 1990; Freed et al., 1991; Ivanoff et al., 1991; Bergeron et al.,1992; Grimaila et al., 1992; Page et al., 1992; Travis et al., 1992) andthe binding of antibodies to this domain (Putney et al., 1986; Goudsmitet al., 1988; Linsley et al., 1988; Rusche et al., 1988; Skinner et al.,Javeherian et al., 1989) have been shown to disrupt a virus entryprocess other than CD4 binding. Accordingly, one can create derivativesand change the phenotype for a particular receptor by substituting V3loops.

One can inhibit infection by directly blocking receptor binding. Thiscan be accomplished by a range of different approaches. For example,antibodies. One preferred approach is the use of antibodies to thebinding site for these chemokine receptors. Antibodies to thesereceptors can be prepared by standard means using the stable immunogenicoligomers. For example, one can use single chain antibodies to targetthese binding sites.

As used herein the inhibition of HIV infection means that as compared toa control situation infection is reduced, inhibited or prevented.Infection is preferably at least 20% less, more preferably at least 40%less, even more preferably at least 50% less, still more preferably atleast 75% less, even more preferably at least 80% less, and yet morepreferably at least 90% less than the control.

One preferred use of the antibodies is to minimize the risk of HIVtransmission. These antibodies can be included in ointments, foams,creams that can be used during sex. For example, they can beadministered preferably prior to or just after sexual contact such asintercourse. One preferred composition would be a vaginal foamcontaining one of the antibodies. Another use would be in systemicadministration to block HIV-1 replication in the blood and tissues. Theantibodies could also be administered in combination with other HIVtreatments.

An exemplary pharmaceutical composition is a therapeutically effectiveamount of a the oligomer, antibody etc. that for examples affects theability of the receptor to facilitate HIV infection or for the DNAsequence or the oligomer that can induce an immune reaction, therebyacting as a prophylactic immunogen, optionally included in apharmaceutically-acceptable and compatible carrier. The term“pharmaceutically-acceptable and compatible carrier” as used herein, anddescribed more fully below, includes (i) one or more compatible solid orliquid filler diluents or encapsulating substances that are suitable foradministration to a human or other animal, and/or (ii) a system, such asa retroviral vector, capable of delivering the molecule to a targetcell. In the present invention, the term “carriers” thus denotes anorganic or inorganic ingredient, natural or synthetic, with which themolecules of the invention are combined to facilitate application. Theterm “therapeutically-effective amount” is that amount of the presentpharmaceutical compositions which produces a desired result or exerts adesired influence on the particular condition being treated. Forexample, the amount necessary to raise an immune reaction to provideproplyactic protection. Typically when the composition is being used asa prophylactic immunogen at least one “boost” will be administered at aperiodic internal after the initial administration. Variousconcentrations may be used in preparing compositions incorporating thesame ingredient to provide for variations in the age of the patient tobe treated, the severity of the condition, the duration of the treatmentand the mode of administration.

The term “compatible”, as used herein, means that the components of thepharmaceutical compositions are capable of being commingled with a smallmolecule, nucleic acid and/or polypeptides of the present invention, andwith each other, in a manner such that does not substantially impair thedesired pharmaceutical efficacy.

Dose of the pharmaceutical compositions of the invention will varydepending on the subject and upon particular route of administrationused. Dosages can range from 0.1 to 100,000 μg/kg per day, morepreferably 1 to 10,000 μg/kg. By way of an example only, an overall doserange of from about, for example, 1 microgram to about 300 microgramsmight be used for human use. This dose can be delivered at periodicintervals based upon the composition. For example on at least twoseparate occasions, preferably spaced apart by about 4 weeks. Othercompounds might be admisnistered daily. Pharmaceutical compositions ofthe present invention can also be administered to a subject according toa variety of other, well-characterized protocols. For example, certaincurrently accepted immunization regimens can include the following: (i)administration times are a first dose at elected date; a second dose at1 month after first dose; and a third dose at 5 months after seconddose. See Product Information, Physician's Desk Reference, Merck Sharp &Dohme (1990), at 1442–43. (e.g., Hepatitis B Vaccine-type protocol);(ii) Recommended administration for children is first dose at electeddate (at age 6 weeks old or older); a second dose at 4–8 weeks afterfirst dose; a third dose at 4–8 weeks after second dose; a fourth doseat 6–12 months after third dose; a fifth dose at age 4–6 years old; andadditional boosters every 10 years after last dose. See ProductInformation, Physician's Desk Reference, Merck Sharp & Dohme (1990), at879 (e.g., Diptheria, Tetanus and Pertussis-type vaccine protocols).Desired time intervals for delivery of multiple doses of a particularcomposition can be determined by one of ordinary skill in the artemploying no more than routine experimentation.

The antibodies, DNA sequences or oligomers of the invention may also beadministered per se (neat) or in the form of a pharmaceuticallyacceptable salt. When used in medicine, the salts should bepharmaceutically acceptable, but non-pharmaceutically acceptable saltsmay conveniently be used to prepare pharmaceutically acceptable saltsthereof and are not excluded from the scope of this invention. Suchpharmaceutically acceptable salts include, but are not limited to, thoseprepared from the following acids: hydrochloric, hydrobromic, sulfuric,nitric, phosphoric, maleic, acetic, salicylic, p-toluene-sulfonic,tartaric, citric, methanesulphonic, formic, malonic, succinic,naphthalene-2-sulfonic, and benzenesulphonic. Also, pharmaceuticallyacceptable salts can be prepared as alkaline metal or alkaline earthsalts, such as sodium, potassium or calcium salts of the carboxylic acidgroup. Thus, the present invention also provides pharmaceuticalcompositions, for medical use, which comprise nucleic acid and/orpolypeptides of the invention together with one or more pharmaceuticallyacceptable carriers thereof and optionally any other therapeuticingredients.

The compositions include those suitable for oral, rectal, intravaginal,topical, nasal, ophthalmic or parenteral administration, all of whichmay be used as routes of administration using the materials of thepresent invention. Other suitable routes of administration includeintrathecal administration directly into spinal fluid (CSF), directinjection onto an arterial surface and intraparenchymal injectiondirectly into targeted areas of an organ. Compositions suitable forparenteral administration are preferred. The term “parenteral” includessubcutaneous injections, intravenous, intramuscular, intrasternalinjection or infusion techniques.

The compositions may conveniently be presented in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.Methods typically include the step of bringing the active ingredients ofthe invention into association with a carrier which constitutes one ormore accessory ingredients.

Compositions of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets, tablets orlozenges, each containing a predetermined amount of the nucleic acidand/or polypeptide of the invention in liposomes or as a suspension inan aqueous liquor or non-aqueous liquid such as a syrup, an elixir, oran emulsion.

Preferred compositions suitable for parenteral administrationconveniently comprise a sterile aqueous preparation of the molecule ofthe invention which is preferably isotonic with the blood of therecipient. This aqueous preparation may be formulated according to knownmethods using those suitable dispersing or wetting agents and suspendingagents. The sterile injectable preparation may also be a sterileinjectable solution or suspension in a non-toxic parenterally-acceptablediluent or solvent, for example as a solution in 1,3-butane diol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose any bland fixed oil may be employedincluding synthetic mono- or diglycerides. In addition, fatty acids suchas oleic acid find use in the preparation of injectibles.

The term “antibodies” is meant to include monoclonal antibodies,polyclonal antibodies and antibodies prepared by recombinant nucleicacid techniques that are selectively reactive with polypeptides encodedby eukaryotic nucleotide sequences of the present invention. The term“selectively reactive” refers to those antibodies that react with one ormore antigenic determinants on e.g. gp120 and do not react with otherpolypeptides. Antigenic determinants usually consist of chemicallyactive surface groupings of molecules such as amino acids or sugar sidechains and have specific three dimensional structural characteristics aswell as specific charge characteristics. Antibodies can be used fordiagnostic applications or for research purposes, as well as to blockbindiner interactions.

For example, cDNA clone encoding a gp120 of the present invention may beexpressed in a host using standard techniques (see above; see Sambrooket al., Molecular Cloning; A Laboratory Manual, Cold Spring HarborPress, Cold Spring Harbor, N.Y.: 1989) such that 5–20% of the totalprotein that can be recovered from the host is the desired protein.Recovered proteins can be electrophoresed using PAGE and the appropriateprotein band can be cut out of the gel. The desired protein sample canthen be eluted from the gel slice and prepared for immunization.Preferably, one would design a stable cell capable of expressing highlevels of the proteins which be selected and used to generate antibodies

For example, mice can be immunized twice intraperitoneally withapproximately 50 micrograms of protein immunogen per mouse. Sera fromsuch immunized mice can be tested for antibody activity byimmunohistology or immunocytology on any host system expressing suchpolypeptide and by ELISA with the expressed polypeptide. Forimmunohistology, active antibodies of the present invention can beidentified using a biotin-conjugated anti-mouse immunoglobulin followedby avidin-peroxidase and a chromogenic peroxidase substrate.Preparations of such reagents are commercially available; for example,from Zymad Corp.

San Francisco, Calif. Mice whose sera contain detectable activeantibodies according to the invention can be sacrificed three days laterand their spleens removed for fusion and hybridoma production. Positivesupernatants of such hybridomas can be identified using the assaysdescribed above and by, for example, Western blot analysis.

To further improve the likelihood of producing an antibody as providedby the invention, the amino acid sequence of polypeptides encoded by aeukaryotic nucleotide sequence of the present invention may be analyzedin order to identify desired portions of amino acid sequence which maybe associated with receptor binding. For example, polypeptide sequencesmay be subjected to computer analysis to identify such sites.

For preparation of monoclonal antibodies directed toward polypeptidesencoded by a eukaryotic nucleotide sequence of the invention, anytechnique that provides for the production of antibody molecules bycontinuous cell lines may be used. For example, the hybridoma techniqueoriginally developed by Kohler and Milstein (Nature, 256: 495–497,1973),as well as the trioma technique, the human B-cell hybridoma technique(Kozbor et al., Immunology Today, 4:72), and the EBV-hybridoma techniqueto produce human monoclonal antibodies, and the like, are within thescope of the present invention. See, generally Larrick et al., U.S. Pat.No. 5,001,065 and references cited therein. Further, single-chainantibody (SCA) methods are also available to produce antibodies againstpolypeptides encoded by a eukaryotic nucleotide sequence of theinvention (Ladner et al. U.S. Pat. Nos. 4,704,694 and 4,976,778).

The monoclonal antibodies may be human monoclonal antibodies or chimerichuman-mouse (or other species) monoclonal antibodies. The presentinvention provides for antibody molecules as well as fragments of suchantibody molecules.

Those of ordinary skill in the art will recognize that a large varietyof possible moieties can be coupled to the resultant antibodies or toother molecules of the invention. See, for example, “ConjugateVaccines”, Contributions to Microbiology and Immunology, J. M. Cruse andR. E. Lewis, Jr (eds), Carger Press, New York, (1989), the entirecontents of which are incorporated herein by reference.

Coupling may be accomplished by any chemical reaction that will bind thetwo molecules so long as the antibody and the other moiety retain theirrespective activities. This linkage can include many chemicalmechanisms, for instance covalent binding, affinity binding,intercalation, coordinate binding and complexation. The preferredbinding is, however, covalent binding. Covalent binding can be achievedeither by direct condensation of existing side chains or by theincorporation of external bridging molecules. Many bivalent orpolyvalent linking agents are useful in coupling protein molecules, suchas the antibodies of the present invention, to other molecules. Forexample, representative coupling agents can include organic compoundssuch as thioesters, carbodiimides, succinimide esters, diisocyanates,glutaraldehydes, diazobenzenes and hexamethylene diamines. This listingis not intended to be exhaustive of the various classes of couplingagents known in the art but, rather, is exemplary of the more commoncoupling agents. (See Killen and Lindstrom 1984, “Specific killing oflymphocytes that cause experimental Autoimmune Myasthenia Gravis bytoxin-acetylcholine receptor conjugates.” Jour. Immun. 133:1335–2549;Jansen, F. K., H. E. Blythman, D. Carriere, P. Casella, O. Gros, P.Gros, J. C. Laurent, F. Paolucci, B. Pau, P. Poncelet, G. Richer, H.Vidal, and G. A. Voisin. 1982. “Immunotoxins: Hybrid molecules combininghigh specificity and potent cytotoxicity”. Immunological Reviews62:185–216; and Vitetta et al., supra).

Preferred linkers are described in the literature. See, for example,Ramakrishnan, S. et al., Cancer Res. 44:201–208 (1984) describing use ofMBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester). See also, Umemotoet al. U.S. Pat. No. 5,030,719, describing use of halogenated acetylhydrazide derivative coupled to an antibody by way of an oligopeptidelinker. Particularly preferred linkers include: (i) EDC(1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride; (ii) SMPT(succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)-toluene(Pierce Chem. Co., Cat. (21558G); (iii) SPDP(succinimidyl-6[3-(2-pyridyldithio) propionamido] hexanoate (PierceChem. Co., Cat #21651G); (iv) Sulfo-LC-SPDP (sulfosuccinimidyl6[3-(2-pyridyldithio)-propianamide] hexanoate (Pierce Chem. Co. Cat.#2165-G); and (v) sulfo-NHS(N-hydroxysulfo-succinimide: Pierce Chem.Co., Cat. #24510) conjugated to EDC.

The linkers described above contain components that have differentattributes, thus leading to conjugates with differing physio-chemicalproperties. For example, sulfo-NHS esters of alkyl carboxylates are morestable than sulfo-NHS esters of aromatic carboxylates. NHS-estercontaining linkers are less soluble than sulfo-NHS esters. Further, thelinker SMPT contains a sterically hindered disulfide bond, and can formconjugates with increased stability. Disulfide linkages, are in general,less stable than other linkages because the disulfide linkage is cleavedin vitro, resulting in less conjugate available. Sulfo-NHS, inparticular, can enhance the stability of carbodimide couplings.Carbodimide couplings (such as EDC) when used in conjunction withsulfo-NHS, forms esters that are more resistant to hydrolysis than thecarbodimide coupling reaction alone.

Antibodies of the present invention can be detected by appropriateassays, such as the direct binding assay discussed earlier and by otherconventional types of immunoassays. For example, a sandwich assay can beperformed in which the receptor or fragment thereof is affixed to asolid phase. Incubation is maintained for a sufficient period of time toallow the antibody in the sample to bind to the immobilized polypeptideon the solid phase. After this first incubation, the solid phase isseparated from the sample. The solid phase is washed to remove unboundmaterials and interfering substances such as non-specific proteins whichmay also be present in the sample. The solid phase containing theantibody of interest bound to the immobilized polypeptide of the presentinvention is subsequently incubated with labeled antibody or antibodybound to a coupling agent such as biotin or avidin. Labels forantibodies are well-known in the art and include radionuclides, enzymes(e.g. maleate dehydrogenase, horseradish peroxidase, glucose oxidase,catalase), fluors (fluorescein isothiocyanate, rhodamine, phycocyanin,fluorescamine), biotin, and the like. The labeled antibodies areincubated with the solid and the label bound to the solid phase ismeasured, the amount of the label detected serving as a measure of theamount of anti-urea transporter antibody present in the sample. Theseand other immunoassays can be easily performed by those of ordinaryskill in the art.

The following Examples serve to illustrate the present invention, andare not intended to limit the invention in any manner.

Specific groups of HIV-1 neutralizing antibodies directed against thegp120 V3 loop or CD4-induced (CD4i) epitopes were able to block thebinding of gp120-sCD4 complexes to CCR5-expressing cells (3,4). The CD4iepitopes are conserved, discontinuous gp120 structures that are exposedbetter after CD4 binding (5). Mutagenic analysis suggested that elementsof the conserved stem of the V1V2 stem-loop and of the fourth conservedregion of gp120 comprise the CD4i epitopes (5). The following examplesdemonstrate that conserved gp120 residues near or within the CD4iepitopes are critical for CCR5 binding.

An assay was established that could assess the CCR5-binding ability of apanel of HIV-1 gp120 glycoprotein mutants. The mutants were created bythe introduction of single amino acid changes in gp120 residues near orwithin regions previously shown to be important for the integrity of theCD4i epitopes (5). The wtΔ glycoprotein, which lacks the V1/V2 variableloops and the N-terminus and is derived from the YU2 primarymacrophage-tropic HIV-1 isolate (7), was the starting point for thestudies (FIGS. 1A-E). This protein was chosen because it had been shownto bind CD4 and CCR5 with high affinity (3,8,9). Furthermore, the use ofthis protein minimized the opportunities for indirect effects of gp120amino acid changes on CCR5 binding (e.g., by repositioning the V1/V2loops, which can mask CD4i epitopes (9)). Metabolically labeled wtΔ andmutant derivatives were produced in 293T cells and incubated with mouseL1.2 cells stably expressing human CCR5 (3), in either the absence orpresence of sCD4. The cells were washed and lysed, and bound gp120protein was detected by precipitation with a mixture of sera from HIV-1infected individuals (10).

The wtΔ protein efficiently bound to the L1.2-CCR5 cells in the presenceof sCD4. Binding was dramatically reduced when sCD4 was not present inthe assay. The wtΔ protein binding to the L1.2-CCR5 cells was inhibitedby preincubation of the wtΔ protein with the 17b antibody. Binding wasalso inhibited by incubation of the L1.2-CCR5 cells with the 2D7antibody against CCR5 (C 11) or with the CCR5 ligand, MIP-1,8 (12). TheC11 antibody, which is directed against a gp120 region dispensable forCCR5 binding (3), did not block the binding of the wtΔ protein to theL1.2-CCR5 cells (data not shown). The wtΔ protein did not bindappreciably to the parental L1.2 cells not expressing CCR5 even in thepresence of sCD4. These results indicate that the wtΔ protein binds CCR5in a specific, CD4-dependent manner.

The binding of the panel of gp120 mutants to the L1.2-CCR5 cells in theabsence and presence of sCD4 was measured. The recognition of the mutantproteins by sCD4 and by monoclonal antibodies that recognizediscontinuous gp120 epitopes (5,13) was assessed in parallel (10).Changes in several gp120 amino acids resulted in dramatic reductions inthe ability of the protein to bind to L1.2-CCR5 cells in the presence ofsCD4 (Table 1). In some cases (257 T/D, 370 E/Q and 383 F/S), theattenuated CD4-binding ability of the mutant proteins could account forthe observed reduction in binding to the L1.2-CCR5 cells. In most cases,however, the mutant proteins that were deficient in CCR5 binding stillbound sCD4 and at least one of the monoclonal antibodies recognizingdiscontinuous gp120 epitopes. As expected, some of the introduced aminoacid changes decreased recognition by the 17b antibody. Interestingly,two of the gp120 amino acid changes (437 P/A, 442 Q/L) resulted in anincrease in CCR5 binding compared with the wtΔ protein, even though CD4binding was not significantly increased. In the absence of sCD4, the 437P/A and 442 Q/L envelope glycoprotein mutants bound to the L1.2-CCR5cells slightly better than the other mutants and the wtΔ protein, whichexhibited very low levels of binding (data not shown).

Table 1. Phenotypes of HIV-1 gp120 mutants. The ability of the wtΔ andmutant glycoproteins to bind CCR5 expressed on L1.2 cells was determined(10). The recognition of the wtΔ and mutant glycoproteins by sCD4 andmonoclonal antibodies was determined (10). All values reported arerelative to those seen for the wtΔ protein. Values represent the averageof at least two independent experiments and exhibited less than 30%variation from the value shown.

Protein (Fractional Solvent Ligand Binding Accessibility)* CCR5 BindingHsCD4 17b CG10 F105 wtΔ 1.00 1.00 1.00 1.00 1.00 107 D/R 1.02 1.02 0.971.11 1.14 114 Q/L 1.22 0.79 0.73 0.71 0.75 117 K/D (0.45) 0.15 0.74 0.640.42 0.83 121 K/D (0.57) 0.07 0.73 0.11 0.0 0.99 122 US 0.98 0.84 1.070.18 1.11 123 T/D (0.49) 0.08 0.99 1.06 0.0 1.25 197 N/D 1.33 1.34 0.800.81 1.11 199 S/L 1.50 1.32 0.94 1.03 1.04 200 V/S 0.84 0.91 1.05 0.491.06 201 I/A 0.46 0.90 0.67 0.84 0.81 203 Q/L 0.68 0.85 0.88 0.52 0.93207 KID (0.23) 0.0 0.85 0.46 0.13 0.98 209 S/L 1.00 1.11 0.85 1.01 1.00210 F/S 0.65 0.81 0.81 0.85 0.74 211 E/K 0.73 1.13 1.03 1.12 1.24 257T/D 0.05 0.0 0.49 0.06 0.0 295 N/E 0.86 0.75 0.73 0.98 0.79 308 N/D 0.311.10 0.89 0.93 1.03 317 US 0.08 1.12 1.05 1.13 1.03 330 H1A 0.22 0.750.55 0.66 0.64 AV3 (/~298–329) 0.0 0.80 0.08 1.27 0.93 370 E/Q 0.17 0.01.04 0.12 0.0 372 V/S 0.85 1.03 1.08 1.09 0.44 373 T/D 0.48 1.12 1.101.16 1.10 377 N/E (0.04) 0.22 0.71 0.52 0.65 0.60 381 E/R (0.07) 0.070.81 0.75 0.29 0.96 383 F/S 0.04 0.0 0.0 0.07 0.0 386 N/D 1.22 1.14 0.970.90 0.97 419 R/D (0.82) 0.19 0.86 0.02 0.48 0.82 420 I/R (0.14) 0.060.59 0.0 0.72 0.72 421 K/D (0.32) 0.07 0.86 0.19 0.0 0.0 422 Q/L (0.35)0.07 0.53 0.0 0.20 0.55 423 I/S 0.61 0.97 0.05 0.30 1.03 424 I/S 0.370.25 0.48 0.83 0.81 426 M/A 0.75 0.69 0.69 0.72 1.11 429 E/R 1.54 1.171.00 1.05 0.82 432 K/A 0.61 1.0 0.92 0.0 1.45 434 MIA 1.22 0.90 0.650.07 1.04 435 Y/S 0.21 0.33 0.22 0.29 1.00 436 A/S 0.98 1.05 0.91 0.991.23 437 P/A 1.79 0.80 0.68 0.78 0.82 438 P/A (0.28) 0.06 1.18 1.00 1.131.18 439 I/A 0.45 0.68 0.76 0.76 0.84 440 R/D (0.43) 0.09 1.03 1.05 1.051.13 441 GN (0.91) 0.0 0.67 0.70 0.62 0.78 442 Q/L 2.00 1.11 0.74 1.050.83 444 RID (0.80) 0.25 0.79 0.67 0.94 0.74 474 D/R 1.03 0.59 0.81 0.740.0 *The number of the mutant wtΔ glycoproteins is based on the sequenceof the prototypic HXBc2 gp120 glycoprotein (24), with 1 representing theinitiator methionine. The wild-type YU2 gp120 residue is listed,followed by the substituted residue. Amino acid abbreviations: A,alanine; D, aspartic acid; E, glutamic acid; F. phenylalanine; G.glycine; H. histidine; I, isoleucine; K, Lysine; L, leucine; M,methionine; N. asparagine; P. proline; Q. glutamine; R. arginine; S.serine; T.threonine; V, valine; Y. tyrosine. The fractional solventaccessibilities associated with gp120 residues in which changesspecifically disrupted CCR5 binding are shown in parentheses. Fractionalsolvent accessibility was calculated as the ratio of solvent-accessiblesurface area for atoms of amino-acid residue X in the gp120 core(without carbohydrate moieties) to the area obtained after reducing thestructure to a Gly-X-Gly tripeptide (24). Values cited are forside-chain atoms exceptfor glycine 441 where the value for all atoms isgiven.

HThe binding of the wtΔ glycoprotein to L1.2-CCR5 cells was shown to belinearly related to the concentration of wtΔ protein in the transfected293T cell supernatants, over the range of concentrations used in theseexperiments. The total amount of wtΔ and mutant glycoprotein present inthe 293T cell supernatants was estimated by precipitation with an excessof a mixture of sera from HIV-1-infected individuals. The amount of wtΔand mutant glycoprotein bound to the L1.2-CCR5 cells was determined asdescribed (10). The value for CCR5 binding was calculated using thefollowing formula:

${{CCR5}\mspace{14mu}{binding}} = {\frac{{Bound}\mspace{14mu}{mutant}\mspace{14mu}{protein}}{{Bound}\mspace{14mu}{wt}\;\Delta\mspace{14mu}{protein}} \times \frac{{Total}\mspace{14mu}{wt}\;\Delta\mspace{14mu}{protein}}{{Total}\mspace{14mu}{mutant}\mspace{14mu}{protein}}}$

The recognition of the wtΔ and mutant glycoproteins by sCD4 andantibodies was determined by precipitation of radiolabeled envelopeglycoproteins in transfected 293T cell supernatants as described (10).In parallel, the labeled envelope glycoproteins were precipitated withan excess of a mixture of sera from HIV-1-infected individuals. Thevalue for ligand binding was calculated using the following formula:

${{Ligand}\mspace{14mu}{binding}} = {\frac{{Mutant}\mspace{14mu}{protein}_{ligand}}{{wt}\;\Delta\mspace{20mu}{protein}_{ligand}} \times \frac{{wt}\;\Delta\mspace{14mu}{protein}_{{serum}\mspace{14mu}{mixture}}}{{Mutant}\mspace{14mu}{protein}_{{serum}\mspace{14mu}{mixture}}}}$

In the sCD4 and 17b columns, the values in bold indicate gp120 residuesthat exhibit decreased solvent accessibility in the presence of thetwo-domain sCD4 or 17b Fab, respectively, in the ternary complex (6).Changes in solvent accessibility were calculated using the MS program ofMichael Connolly.

Graphics. Molecular graphics were produced using Midas-Plus (Universityof California, San Francisco) and GRASP.30

Assignment of variability. Variability in gp120 residues was assessedusing an alignment of sequences derived from approximately 400 HIV-1,HIV-2 and simian immunodeficiency viruses.¹³ Residues were assignedvariability indices and color coded as follows:

-   -   Red: conserved in all primate immunodeficiency viruses;    -   Orange: conserved in all HIV-1, including groups M and 0 and        chimpanzee isolates;    -   Yellow: some variation among HIV-1 isolates (divergence from the        consensus sequence in 1–8 of the 12 HIV-1 groups examined).

Green: variable among HIV-1 isolates (divergence from the consensussequence in. 9 of the 12 HIV-1 groups examined).

Molecular modeling. Residues 88, 89, and 397–409, which are disorderedin the ternary complex crystals (H. Deng et al., Nature, 381:661–666(1996), were built manually using the program TOM. For the V4 loop(residues 397–409), a dominant constraint was the distance between theordered residues 396 and 410 (C—C distance of 26.88 Δ). For thecarbohydrate, examination of the N-linked carbohydrate in severalcrystal structures (e.g. 1fc2, 1gly, 1lte) showed that the core commonto both high-mannose and complex N-linked sugars, (NAG)₂(MAN)₃, did notdiffer greatly in conformation after alignment of the first NAG. Thiscore, which represents roughly half the total glycosylation for atypical N-linked site, was built onto each of the 18 consensus N-linkedglycosylation sites found on the HXBc2 gp120 core. The stereochemistryof this initial model was refined using 4; simulated annealing in XPLOR.Briefly, the model was heated to between 2,500′ and 3,500° K., and “slowcooled” in steps of 25° to 300° K. At each step, molecular dynamics wereperformed with the core gp120 fixed, allowing only the modeled residuesand carbohydrate (including any attached Asn) to move. In three separateruns, performing molecular dynamics for 5 fs/step, all steric clashescould be removed and the geometry idealized, with an average root meansquare (RMS) of carbohydrate movement of only ˜3.5Δ. Four subsequentruns were made using dynamic times of between 50–75 fs/step. Thecarbohydrate positions obtained from these runs differed moresubstantially from those in the starting model (average carbohydrate RMSdifference of roughly 8Δ). Two of the models from these longerannealings were much more similar to each other than to the rest (RMSdifferences in carbohydrate of ˜4Δ versus ˜8Δ for all other models). Onehad been heated to 3,500° K. with dynamics of 75 fs/step. The other(shown in the figures here) was heated to only 2,500° K. with dynamicsof 50 fs/step. In general the RMS movement of the NAG sugars was roughlyhalf the RMS movement of the MAN sugars, reflecting greaterconformational flexibility further from the protein surface.

In primary sequence, human and simian immunodeficiency virus gp120glycoproteins consist of five variable regions (V1–V5) interposed amongmore conserved regions (G. Alkhatib et al., Science 272:1955–1958(1996)). Variable regions V1–V4 form, exposed loops anchored at theirbases by disulfide bonds (L. Wu et al., Nature, 384:179–183 (1996)).Neutralizing antibodies recognize both variable and conserved gp120structures. The V2 and V3 loops contain epitopes for strain-restrictedneutralizing antibodies (E. Emini et al., Nature, 355:728–730 (1992); S.Putney et al., Science, 234:1392–1395 (1986); and C. Bruck et al.,Colloque des Cent Garde, 227–233 (1990). More broadly neutralizingantibodies recognize discontinuous, conserved epitopes in three regionsof the gp120 glycoprotein (Table 2). In HIV-1-infected humans, the mostabundant of these are directed against the CD4 binding site (CD4BS) andblock gp120 CD4 interaction (Y. Feng et al., Science 272:872–877 (1996);and H. Choe et al., Cell, 85:1135–1148 (1996)). Less common areantibodies against epitopes induced or exposed upon CD4 binding (CD4i)(P. Berman et al., Nature, 345:622–625 (1990)). Both CD4i and V3antibodies disrupt the binding of gp120-CD4 complexes to chemokinereceptors (B. Doranz et al., Cell, 85:1149–1158 (1996); and T. Draoic etal., Nature, 381:667–673 (1996)). A third gp120 neutralization epitopeis defined by a unique monoclonal antibody, 2G 12, (W. Robey et al.,Proc. Natl. Acad. Sci. U.S.A., 83:7023–7027 (1986)) which does notefficiently block receptor binding T. Draoic et al., Nature, 381:667–673(1996)).

The X-ray crystal structure of an HIV-1 gp120 core in a ternary complexwith two-domain soluble CD4 and the Fab fragment of the CD4i antibody,17b. The gp120 core lacks the V1/V2 and V3 variable loops, as well as N-and C-terminal sequences, which interact with the gp41 glycoprotein (M.Lu et al., Nature Structural Biol., 2:1075–1082 (1995)), and isenzymatically deglycosylated (H. Deng et al., Nature, 381:661–666(1996); and K. Steimer et al., Science, 254:105–108 (1991)). Despitethese modifications, the gp120 core binds CD4 and antibodies againstCD4BS and CD4i epitopes (K. Steimer et al., Science, 254:105–108 (1991);and M. Posner et al., J. Immunol., 146:4325–4332 (1991)) and thusretains structural integrity. The gp120 core is composed of an innerdomain, an outer domain and a third element, the “bridging sheet” (H.Deng et al., Nature, 381:661–666 (1996)) (FIG. 1 a). All threestructural elements contribute, either directly or indirectly, to CD4and chemokine receptor binding.¹²

Although generally well-conserved compared with the five variableregions, some variability in the surface of the gp120 core is evidentwhen the sequences of all primate immunodeficiency viruses are analyzed.This variability is disproportionately associated with the surface ofthe outer domain proximal to the V4 and V5 regions and removed from thereceptor-binding regions (FIGS. 1C and 2). The A, C, D and E surfaceloops (12) contribute to the variability of this surface. The potentialN-linked glycosylation sites present in the gp120 core are concentratedin this variable half of the protein. In fact, the only conservedresidues apparent on this relatively variable surface are asparagine 356and threonine/serine 358, which constitute a complex carbohydrateaddition site within the E loop. Since most carbohydrate moieties mayappear as “self” to the immune system, the extensive glycosylation ofthe outer domain surface should render it less visible to immunesurveillance. This helps to explain why antibodies directed against thisgp120 surface have been identified so infrequently.

The receptor-binding regions retained in the gp120 core arewell-conserved among primate immunodeficiency viruses (H. Deng et al.,Nature, 381:661–666 (1996)). Also highly conserved is the surface of theinner domain spanned by the β1 helix and located opposite the variablesurface described above. This surface is likely to interact with gp41and/or with N-terminal gp120 segments absent from the gp120 core. Thisinner domain surface and the receptor-binding regions are devoid ofglycosylation. In conjunction with prior mutagenic and antibodycompetition analyses, (A. Pinter et al., J. Virol., 63:2674–2679 (1989);M. Lu et al., Nature Structural Biol., 2:1075–1082 (1995); P. Berman etal., Nature, 345:622–625 (1990); W. Robey et al., Proc. Natl. Acad. Sci.U.S.A., 83:7023–7027 (1986); J. Rusche et al., Proc Natl. Acad. Sci.U.S.A., 85:3198–3202 (1988); and K. Steimer et al., Science, 254:105–108(1991)) the gp120 core structure reveals for the first time the spatialpositioning of the conserved gp120 neutralization epitopes. Although themajor variable loops are either absent (V1/V2 and V3) or poorly resolved(V4) in the gp120 core structure, their approximate positions can bededuced (FIG. 3A). The conserved gp120 neutralization epitopes arediscussed in relation to these variable loops and to the variable,glycosylated core surface.

a) CD4i epitopes. The gp120 epitope recognized by the CD4i antibody,17b, can be directly visualized in the crystallized ternary complex(FIGS. 3B and 3C). Strands from the gp120 fourth conserved (C4) regionand the V1/V2 stem contribute to an antiparallel β-sheet (the “bridgingsheet” (see FIG. 1A)) that contacts the antibody. The vast majority ofgp120 residues previously implicated in formation of the CD4i epitopes¹⁸(Table 2) are located either within this β-sheet or in nearbystructures. With the exception of Thr 202 and Met 434, the gp120residues in contact with the 17b Fab are highly conserved among HIV-1isolates (FIG. 1C, 2 and 3A). The prominent (“male”) CDR3 loop of the17b heavy chain dominates the contacts with gp120, with additionalcontacts through the heavy chain CDR2 (H. Deng et al., Nature,381:661–666 (1996)). Unusually, there are minimal 17b light chaincontacts, leaving a large gap between the gp120 core and most of the 17blight chain surface. In the complete gp120 glycoprotein, this gap islikely occupied by the V3 loop. This is consistent with the position andorientation of the V3 stem on the gp120 core structure (H. Deng et al.Nature, 381:661–666 (1996)), the effect of V3 deletions on the bindingof CD4i antibodies in the absence of soluble CD4 (M. Posner et al., J.Immunol., 146:4325–4332 (1991)), the competition of some V3-directedantibodies with CD4i antibodies A. Pinter et al., J. Virol.,63:2674–2679 (1989)), and the ability of both antibody groups to blockchemokine receptor binding (B. Doranz et al., Cell, 85:1149–1158 (1996);and T. Draoic et al., Nature, 381:667–673 (1996). The chemokinereceptor-binding region of gp120 likely consists of elements near orwithin the “bridging sheet” and the V3 loop.

The V2 loop likely resides on the side of the 17b epitope opposite theV3 loop (FIG. 3A). The V1/V2 loops, which vary from 57 to 86 residues inlength,¹³ are dispensable for HIV-1 replication (M. Posner et al., J.Immunol., 146:4325–4332 (1991)); and R. Wyatt et al., J. Virol.,69:5723–5733 (1995)) but decrease the sensitivity of viruses toneutralization by antibodies against V3 and CD4i epitopes (R. Wyatt etal., J. Virol., 69:5723–5733 (1995)). The latter effect is mediatedprimarily by the V2 loop, (M. Posner et al., J. Immunol., 146:4325–4332(1991)) suggesting that part of the V2 loop folds back along the V1/V2stem to mask the “bridging sheet” and adjacent V3 loop. The proximity ofthe V2 and V3 loops is supported by the observation that, in monkeysinfected with simian-human immunodeficiency viruses (SHIVs),neutralizing antibodies are raised against discontinuous epitopes withV2 and V3 components (B. Etemad-Moghadam and J. Sodroski). The CD4iepitopes are apparently masked by the flanking V2 and V3 loops,requiring the evolution of antibodies with protruding (“male”) CDRs toaccess these conserved epitopes. CD4 binding has been suggested toreposition the V1/V2 loops, thus exposing the CD4i epitopes (M. Posneret al., J. Immunol., 146:4325–4332 (1991)). The presence of contactsbetween the V1/V2 stem and CD4 in the crystal structured is consistentwith this model.

b) CD4BS epitopes. CD4 makes a number of contacts within a recessedpocket on the gp120 surface. The gp120-CD4 interface includes twocavities, one water-filled and bounded equally by both proteins, theother extending into the gp120 interior and contacting CD4 only atphenylalanine 43 (H. Deng et al., Nature, 381:661–666 (1996)). Tables 1,2 and FIGS. 3B and 3C show the gp120 residues implicated in theformation of CD4BS epitopes recognized by eight representativeantibodies. CD4BS epitopes are uniformly disrupted by changes in Asp 368and Glu 370, (J. Rusche et al., Proc. Natl. Acad. Sci. U.S.A.,85:3198–3202 (1988)) which surround the opening of the “Phe 43 cavity”.These residues are located on a ridge at the intersection of the tworeceptor-binding gp120 surfaces, consistent with competition studiessuggesting that CD4BS epitopes overlap both the CD4i epitopes and thebinding site for CD4 (A. Pinter et al., J. Virol., 63:2674–2679 (1989);and P. Berman et al., Nature, 345:622–625 (1990)). The location of thegp120 residues implicated in the formation of the CD4BS epitopessuggests that important elements of the CD4-binding surface of gp120 areaccessible to antibodies.

Some CD4BS antibodies, like IgG1b12, are particularly potent atneutralizing HIV-1 (J. Robinson et al., AIDS Res. Hum. Retro, 6:567–580(1990)). IgG1b12 binding is disrupted by gp120 changes that affect thebinding of other CD4BS antibodies but, atypically, is sensitive tochanges in the V1/V2 stem-loop structured The observation that somewell-conserved residues in the gp120 V1/V2 stem contact CD4 (H. Deng etal., Nature, 381:661–666 (1996)) raises the possibility that thisprotruding structure also contributes to the IgG1b12 epitope. This mightincrease the ability of the antibody to access the assembled envelopeglycoprotein trimer, thus increasing neutralizing capability.

While the CD4BS epitopes and the CD4-binding site overlap, severalobservations demonstrate that the binding of CD4BS antibodies differsfrom that of CD4. Changes in Trp 427, a gp120 residue that contacts boththe “Phe 43 cavitv” and CD4, uniformly disrupt CD4 binding but affectthe binding of only some CD4BS antibodies (Table 2). Conversely, somechanges in other cavity-lining gp120 residues, Ser 256 and Thr 257,affect the binding of CD4BS antibodies more than the binding of CD4 (J.Rusche et al., Proc. Natl. Acad. Sci U.S.A., 85:3198–3202 (1988)). Sincethe recessed position of Ser 256 and Thr 257 in the current crystalstructure (FIGS. 3B and 3C) makes direct contacts with antibodyunlikely, either the effects of changes in these residues are indirector the CD4BS antibodies recognize a gp120 conformation that differs fromthe CD4-bound state. With respect to the latter possibility, several ofthe residues implicated in the integrity of the CD4BS epitopes arelocated in the interface between the inner and outer gp120 domains.CD4BS antibodies might recognize a gp120 conformation in which thespatial relationship between the domains is altered compared with theCD4-bound state, thus allowing better surface exposure of theseresidues. Differences between the CD4BS epitopes and the CD4-bindingsite create opportunities for neutralization escape (J. Rusche et al.,Proc. Natl. Acad. Sci. U.S.A., 85:3198–3202 (1988)). The gp120 residuessurrounding the “Phe 43” cavity are highly conserved among primateimmunodeficiency viruses (FIG. 3A), but the observed modest variation inadjacent surface-accessible residues (e.g., Pro 369, Thr 373 and Lys432) could account for decreased recognition of the gp120 glycoproteinfrom some geographic clades of HIV-1 by CD4BS antibodies (S. Tilley etal., Res. Virol., 142:247–259 (1991)). Additional potential forvariation near or within the CD4BS epitopes is created by the unusualwater-filled cavity in the gp120-CD4 binding interface, since CD4binding can apparently tolerate change in the gp120 residues contactingthis cavity (H. Deng et al., Nature, 381:661–666 (1996)).

The recessed nature of the CD4 binding pocket on gp120 (FIG. 1B) candelay the generation of high-affinity antibodies against the CD4BSepitopes and may afford opportunities to minimize the antviral efficacyof such antibodies once they are elicited. The degree of recession isprobably much greater on the full-length, glycosylated gp120 than isevident on the crystallized gp120 core. The recessed pocket is flankedon one side by the V1/V2 stem-loop structure. The characterization ofHIV-1 escape mutants from the IgG1b12 CD4BS antibody and the mapping ofseveral V2 conformational epitopes support a model in which the V2 loopfolds back along the V1/V2 stem, with V2 residues 183–188 proximal toAsp 368 and Glu 370. This model is consistent with observations thatV1/V2 changes, in combination with V3 changes, can alter the exposure ofthe adjacent CD4BS epitopes, particularly on the assembled trimer (R.Wyatt et al., J. Virol., 67:4557–4565 (1993)). The high temperaturefactors associated with the V1/V2 stems imply flexibility in thisprotruding element, expanding the potential range of space occupied bythe V1/V2 stem-loop structure. This could enhance masking of theadjacent CD4BS and CD4i gp120 epitopes and divert antibody responsestowards the variable loops.

Glycosylation can modify the interaction of antibodies with CD4BSepitopes. The D loop, on the rim of the CD4-binding pocket opposite theV1/V2 stem, contains a well-conserved glycosylation site, asparagine276. Changes in this site and at the adjacent alanine 281 have beenassociated with escape from the neutralizing activity of patient sera(D. Ho et al., J. Virol., 65:489–493 (1991)) and have been seen in SHIVsextensively passaged in monkeys (M. Thali et al., J. Virol., 67:39783988(1993)). Another conserved glycosylation site at asparagine 386 liesadjacent to both CD4BS and CD4i epitopes (FIG. 1D) and could diminishantibody responses against those sites. Additionally, in various HIV-1strains, carbohydrates are added to the V2 loop segment (residues186–188) thought to be proximal to the CD4BS epitopes.

c) The 2G12 epitope. The integrity of the 2G12 epitope is disrupted bychanges in gp120 glycosylation, either by glycosidase treatment ormutagenic alteration of specific N-linked carbohydrate addition sites(W. Robey et al., Proc. Natl. Acad. Sci. U.S.A., 83:7023–7027 (1986)).These sites are located on the relatively variable surface of the gp120outer domain, opposite to and approximately 25 Δ away from the CD4binding site (FIGS. 1E, 3B and 3C). The gp120 glycoprotein synthesizedin mammalian cells exhibits a dense concentration of high-mannose sugarsin this region (FIG. 3A). Even in the enzymatically deglycosylated gp120core, carbohydrate residues constitute much of this surface. 2G12 likelybinds at least in part to these carbohydrates, explaining the surprisingconservation of the 2G12 epitope despite the variability of theunderlying protein surface, which includes the stem of the V3 loop andthe V4 variable region. The inclusion of carbohydrate in the epitopemight also explain the apparent rarity with which these antibodies aregenerated. The localization of the 2G12 epitope is consistent withprevious studies indicating that 2G12 forms a unique competition group(A. Pinter et al., J. Virol., 63:2674–2679 (1989); and W. Robey et al.,Proc. Natl. Acad. Sci. U.S.A., 83:7023–7027 (1986)) and does notinterfere with the binding of monomeric gp120 to either CD4 or chemokinereceptors (T. Draoic et al., Nature, 381:667–673 (1996)). Since the 2G12 epitope is predicted to be oriented towards the target cell upon CD4binding (see below), the antibody may sterically impair interactions ofthe oligomeric envelope glycoprotein complex with host cell moieties.

Possible orientations of the exterior glycoproteins in the trimer aresignificantly constrained by the requirement that observed and deducedbinding sites for receptors and neutralizing antibodies, sites ofN-linked glycosylation, and variable structures be exposed on thesurface of the assembled complex. The two-domain CD4 in the ternarycomplex structure was aligned to the structure of four-domain CD429 toorient the trimer model with respect to the target cell membrane. Theconsequences of such a model, which is shown in FIG. 4, are: a) thechemokine receptor-binding sites are clustered at the vertex of thetrimer predicted to be closest to the target cell; b) both variable andconserved neutralization epitopes are concentrated on the half of gp120facing the target cell; c) possibilities for intersubunit interactionsamong the variable structures that could help mask conservedneutralization epitopes are created; d) the subset of gp120glycosylation sites to which complex carbohydrates are added inmammalian cells (L. Wu et al., Nature, 384:179–183 (1996)) iswell-exposed on the outer periphery of the trimer; e) the highlyconserved surface near the β1 helix is available for gp41 and/or gp120protein interactions within the trimers; and f) the surface of theassembled envelope glycoprotein complex is roughly hemispherical, thusminimizing the surface area of the viral spike that is potentiallyexposed to antibodies.

In summary, the X-ray crystal structure of the gp120 core/two-domainCD4/17b Fab complex provides a framework for visualizing keyinteractions between HIV-1 and the humoral immune system. Previousantibody competition analyses suggested that the gp120 surface buried inthe assembled trimer elicits non-neutralizing antibodies. By contrast,the binding sites for neutralizing antibodies cluster on a differentgp120 surface. Our structural studies disclose the existence ofnon-neutralizing and neutralizing faces of gp120, and reveal another,immunologically “silent” face of the glycoprotein (FIG. 3D). This outerdomain surface, along with the major variable loops, contributes to thelarge fraction of the gp120 surface that is protected against antibodyresponses by a dense array of carbohydrates and by the capacity forvariation. The conserved receptor-binding regions of gp120 representattractive targets for immune intervention. However, the elicitation ofantibodies against these conformation-dependent structures has proveninefficient. Since the gp120 epitopes near the receptor-binding regionsspan the inner and outer domains, in terdomain conformational shifts maydecrease their representation in the immunogen pool. The recessed natureof the CD4-binding site likely contributes to its poor immunogenicity.The sequential recognition of two receptors by primate immunodeficiencyviruses allows the conserved elements of the chemokine receptor-bindingsite to be created or exposed by the modified polypeptides describedherein.

TABLE 2 Conserved Epitopes for Neutralizing Antibodies Identified on thegp120 Core Examples of Probable Mechanism Competition Monoclonal ofVirus Group Antibodies gp120 Amino Acids^(b) NeutralizationCharacteristics Selected References CD4-Binding F105 Asn88 (13), Asp113(50), Interference with CD4BS anti- Y. Feng et al., Science, Site 15eLys117 (25), Ser256 (75), gp120-CD4 binding bodies complete 272:872–877(1996); (CD4BS) 21h Thr257 (75), Asn262 (63), with CD4 and H. Choe etal., Cell, 1125h Ala266 (13), Asp368 (100), with antibodies 85:1149–1158(1996); 448D Glu370 (100), Tyr384 (13), against CD4i J. Rusche et al.,Proc. 39.3 Lys421 (50), Trp427 (25), epitopes Natl. Acad. Sci. USA,IgG1b12 Asp457 (13), Pro470 (25), 85:3198–3202 (1988) 830D Asp474 (13),Met475 (13), Asp477 (63), Asp/Leu/Tyr 482/483/484 (25) CD4-induced 17bAsn88, Lys117, Lys121, Interference with CD4 binding P. Berman et al.,Nature, Epitopes 48d Lys207, Ser256, Thr257, chemokine receptorincreases expo- 345:622–625 (1990) (CD4i) Asn262, ΔV3, Glu370, bindingsure of the Glu381, Phe382, Arg419, epitopes as a Ile420, Lys421,Gln422, result of move- Ile423, Trp427, Tyr435, ment of the V2 Pro438,Met475 variable loop 2G12 2G12 Asn295, Thr297, Ser334, Unknown Antibodybind- W. Robey et al., Proc. Asn386, Asn392, Asn397 ing is Natl. Acad.Sci. USA, dependent 83:7023–7027 (1986) upon proper N- linkedglycosyla-tion ^(a)The gp120 competition groups are defined as inReference 5. ^(b)The gp120 amino acids are numbered according to thesequence of the HXBc2 (IIIB) gp120 glycoprotein, where residue 1 is themethionine at the amino-terminus of the signal peptide. Changes in theamino acids listed resulted in significant reduction in antibody bindingto the gp120 glycoprotein (Ref. 18-20). The numbers in parenthesesindicate the percentage of the CD4BS antibodies examined whose bindingis decreased by changes in the indicated residue.

REFERENCES

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All references described herein are incorporated herein by reference.

1. A modified gp120 polypeptide comprising portions of at least twoconserved regions of an envelope protein selected from a the group oflentiviruses consisting of HV-1, HIV-2 and SIV, wherein at least one ofthe following changes relative to the wild-type gp120 protein is made:(a) introduction of disulfide bonds to decrease the free energy offolding relative to the wild type gp120 protein; (b) filling a cavity ofthe gp120 protein with hydrophobic amino acid residues; or (c)introducing a Pro residue at a defined turn structure; wherein themodified polypeptide maintains the overall 3-dimensional structure of adiscontinuous conserved epitope of the wild-type gp120, wherein thediscontinuous conserved epitope is a CD4BS epitope, CD4i epitope or 2G12epitope.
 2. The modified gp120 polypeptide of claim 1, wherein thediscontinuous conserved epitope is a CD4BS epitope or CD4i epitope. 3.The modified gp120 polypeptide of claim 1, wherein the gp120 protein isHIV-1.
 4. The modified gp120 polypeptide of claim 3, wherein disulfidebonds are introduced between at least one of the groups of amino acidsthat correspond to Pro118-Ala443, Leu122-Gly431, Phe210-Gly30, orSer256-Phe376 of the HIV-1 HXBc2 strain.
 5. The modified gp120polypeptide of claims 3 or 4, wherein at least one amino acid residuecorresponding to wild-type gp120 Ser375, Val255, Arg273, Ser481, Ser447,Asn377 of the HIV-1HXBc2 strain, Thr283, or Asp477 of the HIV-1 HXBc2strain, has been substituted with a hydrophobic amino acid residue. 6.The modified gp120 polypeptide of claim 5, wherein at least one of thefollowing amino acid substitutions is present: Trp for Ser375, Val255 orArg 273; Phe for Ser481; Ile for Ser447 or Thr283; Or Leu for Asn377 orThr283.
 7. The modified gp120 polypeptide of claim 5, wherein a Proresidue has been introduced at a defined turn structure.
 8. The modifiedgp120 polypeptide of claim 4, wherein a Pro residue has been introducedat a defined turn structure.
 9. The modified gp120 polypeptide of claim3, wherein a Pro residue has been introduced at a defined turnstructure.
 10. The modified gp120 polypeptide of claim 7, wherein a Proresidue has been substituted for Ile423.
 11. The modified gp120polypeptide of claim 8, wherein a Pro residue has been substituted forIle423.
 12. The modified gp120 polypeptide of claim 9, wherein Pro hasbeen substituted for Ile423.
 13. The modified gp120 polypeptide of claim1, wherein at least two of the changes have been made.
 14. The modifiedgp120 polypeptide of claim 1, wherein at least three of the changes havebeen made.
 15. The modified gp120 polypeptide of claim 1 wherein thecavity of the gp120 protein corresponds to Phe43 of the wild type HIV-1,HXBc2 strain.